WO2018213631A1 - Nano-enabled immunotherapy in cancer - Google Patents

Nano-enabled immunotherapy in cancer Download PDF

Info

Publication number
WO2018213631A1
WO2018213631A1 PCT/US2018/033265 US2018033265W WO2018213631A1 WO 2018213631 A1 WO2018213631 A1 WO 2018213631A1 US 2018033265 W US2018033265 W US 2018033265W WO 2018213631 A1 WO2018213631 A1 WO 2018213631A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
drug carrier
ido
conjugated
methyl
Prior art date
Application number
PCT/US2018/033265
Other languages
French (fr)
Inventor
E. Andre NEL
Huan MENG
Jianqin LU
Original Assignee
The Regents Of The University Of California
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 The Regents Of The University Of California filed Critical The Regents Of The University Of California
Priority to AU2018269742A priority Critical patent/AU2018269742B2/en
Priority to EP18803111.6A priority patent/EP3624810A4/en
Priority to CA3063932A priority patent/CA3063932A1/en
Publication of WO2018213631A1 publication Critical patent/WO2018213631A1/en
Priority to US16/687,368 priority patent/US11433143B2/en
Priority to US17/823,044 priority patent/US20230241244A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4245—Oxadiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/34—Copper; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle

Definitions

  • Pancreatic ductal adenocarcinoma is an almost uniformly fatal disease with a 5-year survival outcome of less than 6% (American Cancer Society. Cancer Facts & Figures (2014). Atlanta: American Cancer Society).
  • PTX paclitaxel
  • irinotecan delivery has had some survival impact (Frese et al. 92012) Cancer Discov. 2(3): 260-269; Passero etal. (2016) Exp. Rev. Anticancer Therap., 16(7): 697-703).
  • PTX delivery by an albumin-nanocarrier can suppress the drug-resistant tumor stroma, allowing increased gemcitabine uptake
  • the delivery of irinotecan by a liposome can improve drug pharmacokinetics.
  • our own studies using mesoporous silica nanoparticles (MSNP) have shown in a robust orthotopic PDAC animal model that it is possible, in one formulation, to include smart-design features to improve irinotecan loading efficacy, carrier stability and safety over a commercial liposomal equivalent, while a second approach was to develop a ratiometric-designed drug carrier for contemporaneous and synergistic delivery of PTX and gemcitabine (Liu et al. (2016) ACS Nano, 10(2): 2702-2715; Meng et al. (2015) ACS Nano, 9(4): 3540-3557).
  • ICD induced cell death
  • CRT calreticulin
  • HMGB-1 a TLR-4 ligand
  • ATP a signal that activates the RLP3
  • ICD will allow more predictable induction of an immune replete status to allow receptor-mediated blockade or perturbation of other immune surveillance pathways to induce durable anti-tumor immunity, which also takes care of metastases.
  • ICD can strengthen the effect of immune checkpoint blocking antibodies as well as indoleamine 2, 3 -di oxygenase (IDO) inhibitors that interfere in this metabolic immune surveillance pathway.
  • ICD provides a deliberate means of initiating and immune "hot" start for subsequent response boosting by metabolic and immune checkpoint inhibitors.
  • the IDO pathway is a relevant metabolic immune checkpoint pathway in breast cancer (and other cancers) because of its overexpression at the tumor site.
  • IDO-1 is the first and rate-limiting enzymatic step in the catabolism of tryptophan in the kynurenine pathway, and exerts potent immunosuppressive effects as a result of the metabolic disturbance of the amino acid ratios (see, e.g., Prendergast et al. (2017) Cane. Res., 77(24): 6795-6811; Lob et al. (2009) Nat. Rev. Cancer, 9: 445-452).
  • IDO IDO effector pathway to control the activity of the mTOR pathway (T-cell activation); activation of the aryl hydrocarbon receptor (AhR) pathway; activation of GCN2 (general control nondereressible), a serine/threonine-protein kinase that senses amino acid deficiency; and development of Tregs.
  • AhR aryl hydrocarbon receptor
  • GCN2 general control nondereressible
  • GCN2 general control nondereressible
  • Tregs serine/threonine-protein kinase that senses amino acid deficiency
  • IDO exerts strong immunosuppressive effects in the TME and regional lymph nodes, culminating in T-cell anergy, decreased cytotoxic killing, and increased accumulation of Tregs at the tumor site (Prendergast et al. (2014) Cancer Immunol. Immunother. 63 : 721-735; Lob et al. (2009) Nat. Rev. Cancer, 9: 445-452).
  • IDO pathway inhibitors a.k.a. indoximod
  • IND 1-methyl-tiyptophan
  • ICD inducers e.g., doxirubicin
  • this goal was accomplished by synthesizing an IDO pathway inhibitor prodrug where the IDO inhibitor (e.g., indoximod) was conjugated to a moietiy (e.g., a phosopholipid) that can be assembled into a lipid bilayer which can in turn be incorporated into a drug delivlery vehicle (e.g 3 a liposome).
  • a moietiy e.g., a phosopholipid
  • IND a phospholipid- conjugated prodrug that self-assembles into a nanovesicle (liposome).
  • an ICD inducer e.g., DOX
  • a doxorubicin (DOX) encapsulating nanocarrier provides a more potent ICD stimulus than the free drug, and can do so synergistically with a small molecule inhibitor (indoximod) of the IDO-1 pathway.
  • the nanocarrier is capable of facilitating this task, in part, by improving the PK of DOX and indoximod (IND) at the tumor site.
  • next generation nanocarrier providing an ICD stimulus and an IDO inhibitor as a promising synergistic immunotherapy platform for BC, including triple negative BC (TNBC) (most responsive to immune checkpoint inhibitors) as well as ER- positive tumors (numerically the largest BC subtype responsible for mortality) and other cancers (e.g., PDAC, and the like).
  • TNBC triple negative BC
  • ER- positive tumors numbererically the largest BC subtype responsible for mortality
  • other cancers e.g., PDAC, and the like.
  • compositions and methods are provided for systemic and/or for local (peri- or intratumoral) delivery of one or more ICD-inducing agents (e.g., doxirubicin, oxaliplatin, etc.) in conjunction with delivery of an inhibitor of the IDO pathway (e.g., indoximod).
  • ICD-inducing agents e.g., doxirubicin, oxaliplatin, etc.
  • an inhibitor of the IDO pathway e.g., indoximod
  • the inhibitor of the IDO inhibitor is conjugated to a nanovesicle-forming moiety (e.g., containing a phospholipid bilayer).
  • compositions and methods are provided where an ICD-inducing agent (e.g., oxaliplatin, doxirubicin, etc.) and an IDO inhibiting agent (e.g., an IDO inhibitor - prodrug) are integrated into a nanocarrier, that allows systemic delivery to a cancer site.
  • an ICD-inducing agent e.g., oxaliplatin, doxirubicin, etc.
  • an IDO inhibiting agent e.g., an IDO inhibitor - prodrug
  • compositions and methods are provided for the treatment or prevention of a cancer via vaccination (e.g., subcutaneous vaccination), utilizing certain cancer cells (e.g., drug-treated cancer cells) in which ICD has been induced ex vivo. It was discovered that vaccination with dying cells initiates a systemic immune response that prevents tumor re-growth.
  • Embodiment 1 A nanovesicle drug carrier for the combined delivery of an
  • nanovesicle drug carrier comprising:
  • a lipid vesicle wherein said lipid vesicle comprises a lipid effective to form a vesicle comprising a lipid bilayer in an aqueous solution, and the lipid bilayer is associated with an inhibitor of the indoleamine 2,3 -di oxygenase (IDO) pathway (IDO pathway inhibitor); and
  • a cargo within said vesicle where said cargo comprises an agent that induces immunogenic cell death (ICD) (ICD-inducer).
  • ICD immunogenic cell death
  • the IDO pathway inhibitor can be conjugated to a component tof the lipid bilayer forming the vesicle.
  • the IDO pathway inhibitor is incorporated into the lipid bilayer (e.g., via alcohol injection, or other methods).
  • Embodiment 2 The nanovesicle drug carrier of embodiment 1, wherein the
  • Embodiment 3 The nanovesicle drug carrier according to any one of embodiments 1-2, wherein said drug carrier, when administered systemically, delivers an amount of an ICD inducer effective to induce or to facilitate induction of immunogenic cell death of a cancer cell at a tumor site.
  • Embodiment 4 The nanovesicle drug carrier according to any one of embodiments 1-3, wherein said drug carrier, when administered systemically, delivers an amount of an IDO pathway inhibitor to partially or fully inhibit the IDO enzyme or IDO pathway at a cancer site.
  • Embodiment 5 The nanovesicle drug carrier according to any one of embodiments 1-4, wherein said IDO pathway inhibitor comprises an inhibitor of the IDO enzyme.
  • Embodiment 6 The nanovesicle drug carrier according to any one of embodiments 1-5, wherein said IDO pathway inhibitor comprises an inhibitor of the IDO pathway downstream from said IDO enzyme.
  • Embodiment 7 The nanovesicle drug carrier according to any one of embodiments 1-6, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l-methyl-tiyptophan (L-1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tiyptophan (L-1MT),
  • said IDO pathway inhibitor comprises an agent selected from the group consisting of of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l-methyl-tiyptophan (L-1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tiyptophan (L-1MT),
  • methylthiohydantoin-dl -tryptophan MTH-Trp, Necrostatin
  • ⁇ -carbolines e.g., 3-butyl-P- carboline
  • Naphthoquinone-based e.g., annulin-B
  • S-allyl-brassinin S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S- methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl- brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol
  • NSC401366 l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDO 1 -derived peptide, NLG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole.
  • Embodiment 8 The nanovesicle drug carrier of embodiment 7, wherein said
  • IDO pathway inhibitor comprises 1 -methyl -tryptophan.
  • Embodiment 9 The nanovesicle drug carrier of embodiment 8, wherein said
  • IDO pathway inhibitor comprises a "D" enantiomer of 1 -methyl -tryptophan (indoximod, 1- MT).
  • Embodiment 10 The nanovesicle drug carrier of embodiment 8, wherein said
  • IDO pathway inhibitor comprises an "L” enantiomer of 1-methyl-tiyptophan (L-MT).
  • Embodiment 1 1 The nanovesicle drug carrier according to any one of embodiments 1-10, wherein said IDO pathway inhibitor, is disposed in a lipid comprising said vesicle and/or conjugated to a lipid comprising said vesicle.
  • Embodiment 12 The nanovesicle drug carrier according to any one of embodiments 1-10, wherein said vesicle comprises a phospholipid and/or a phospholipid prodrug.
  • Embodiment 13 The nanovesicle drug carrier of embodiment 12, wherein said vesicle comprises a phospholipid, and cholesterol (CHOL).
  • said vesicle comprises a phospholipid, and cholesterol (CHOL).
  • Embodiment 14 The nanovesicle drug carrier according to any one of embodiments 12-13, wherein said phospholipid comprises a saturated fatty acid with a C14- C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains.
  • Embodiment 15 The nanovesicle drug carrier of embodiment 14, wherein said phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC),
  • DSPC distearoylphosphatidylcholine
  • DAPC diactylphosphatidylcholine
  • Embodiment 16 The nanovesicle drug carrier of embodiment 14, wherein said phospholipid comprises a natural lipid selected from the group consisting of egg phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC).
  • egg PC egg phosphatidylcholine
  • soy phosphatidylcholine soy phosphatidylcholine
  • Embodiment 17 The nanovesicle drug carrier of embodiment 14, wherein said phospholipid comprises an unsaturated fatty acid selected from the group consisting of l,2-dimyristoleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine,l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dieicosenoyl-sn- glycero-3 -phosphocholine.
  • DOPC 1,2-dieicosenoyl-sn- glycero-3 -phosphocholine.
  • Embodiment 18 The nanovesicle drug carrier according to any one of embodiments 12-17, wherein said vesicle comprises an mPEG phospholipid with a phospholipid C14-C18 carbon chain, and a PEG molecular weight ranging from about 350 Da to 5000 Da.
  • Embodiment 19 The nanovesicle drug carrier of embodiment 18, wherein said vesicle comprises l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (DSPE-PEG).
  • DSPE-PEG l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG
  • Embodiment 20 The nanovesicle drug carrier of embodiment 19, wherein said vesicle comprises DPSE-PEG 2K .
  • Embodiment 21 The nanovesicle drug carrier according to any one of embodiments 1-20, wherein said IDO pathway inhibitor is conjugated to a component of said vesicle.
  • Embodiment 22 The nanovesicle drug carrier of embodiment 21, wherein said IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid.
  • Embodiment 23 The nanovesicle drug carrier according to any one of embodiments 21-22, wherein said IDO pathway inhibitor is conjugated directly to said moiety.
  • Embodiment 24 The nanovesicle drug carrier according to any one of embodiments 21-22, wherein said IDO pathway inhibitor is conjugated to said moiety via a linker.
  • Embodiment 25 The nanovesicle drug carrier according to any one of embodiments 21-22, wherein said IDO pathway inhibitor is conjugated to PGHP.
  • Embodiment 26 The nanovesicle drug carrier according to any one of embodiments 21-24, wherein said IDO pathway inhibitor is conjugated to vitamin E.
  • Embodiment 27 The nanovesicle drug carrier according to any one of embodiments 21-24, wherein said IDO pathway inhibitor is conjugated to cholesterol
  • Embodiment 28 The nanovesicle drug carrier according to any one of embodiments 21-24, wherein said IDO pathway inhibitor is conjugated to a fatty acid.
  • Embodiment 29 The nanovesicle drug carrier of embodiment 28, wherein said IDO pathway is conjugated to oleic acid or docosahexaenoic acid.
  • Embodiment 30 The nanovesicle drug carrier of embodiment 28, wherein said IDO pathway is conjugated to oleic acid or docosahexaenoic acid via an
  • Embodiment 31 The nanovesicle drug carrier according to any one of embodiments 21-24, wherein said IDO pathway inhibitor is conjugated to a lipid.
  • Embodiment 32 The nanovesicle drug carrier of embodiment 31, wherein said IDO pathway inhibitor is conjugated to a phospholipid comprising said lipid vesicle, said phospholipid thereby forming a phospholipid prodrug.
  • Embodiment 33 The nanovesicle drug carrier of embodiment 32, wherein said phospholipid prodrug comprises l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PL).
  • PL l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine
  • Embodiment 34 The nanovesicle drug carrier of embodiment 33, wherein said hospholipid prodrug comprises the structure:
  • Embodiment 35 The nanovesicle drug carrier of embodiment 34, wherein the
  • 1 -methyl -tryptophan component of said conjugated IDO pathway inhibitor comprises a "D" isom r of 1 methyl-tyrptophan (indoximod) characterized by the formula:
  • Embodiment 36 The nanovesicle drug carrier of embodiment 34, wherein the 1 -methyl -tryptophan component of said conjugated IDO pathway inhibitor comprises an "L" isomer of 1 methyl-tyrptophan (L-1MT) characterized by the formula:
  • Embodiment 37 The nanovesicle drug carrier of embodiment 34, wherein the
  • 1 -methyl -tryptophan component of said conjugated IDO pathway inhibitor comprises a mixture of "D” and “L” isomers of 1 -methyl -tryptophan.
  • Embodiment 38 The nanovesicle drug carrier according to any one of embodiments 34-37, wherein said vesicle comprises IND-PL/Chol/DSPE-PEG.
  • Embodiment 39 The nanovesicle drug carrier of embodiment 38, wherein said vesicle comprises about 75% IND-PL, about 20% cholesterol, and about 5% DSPE- PEG 2K .
  • Embodiment 40 The nanovesicle drug carrier according to any one of embodiments 1-39, wherein said ICD inducer comprises a chemotherapeutic agent selected from the group consisting of doxorubicin, oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
  • a chemotherapeutic agent selected from the group consisting of doxorubicin, oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
  • Embodiment 41 The nanovesicle drug carrier of embodiment 40, wherein said ICD inducer comprises doxorubicin.
  • Embodiment 42 The nanovesicle drug carrier drug carrier according to any one of embodiments 1-41, wherein said carrier is colloidally stable.
  • Embodiment 43 The nanovesicle drug carrier according to any one of embodiments 1-42, wherein when the cargo in the nanocarrier is a weak base, and said carrier comprises a cargo-trapping agent.
  • Embodiment 44 The nanovesicle drug carrier of embodiment 43, wherein said cargo trapping agent before reaction with the cargo drug loaded in the vesicle, is selected from the group consisting of triethylammonium sucrose octasulfate (TEA 8 SOS), ( EL ⁇ SC ⁇ , an ammonium salt, a trimethylammonium salt, and a triethylammonium salt.
  • TAA 8 SOS triethylammonium sucrose octasulfate
  • EL ⁇ SC ⁇ an ammonium salt
  • a trimethylammonium salt a trimethylammonium salt
  • a triethylammonium salt a triethylammonium salt.
  • Embodiment 45 The nanovesicle drug carrier according to any one of embodiments 43-44, wherein said cargo-trapping agent before reaction with said drug is ammonium sulfate.
  • Embodiment 46 The nanovesicle drug carrier according to any one of embodiments 1-45, wherein said drug carrier is conjugated to a moiety selected from the group consisting of a targeting moiety, a fusogenic peptide, and a transport peptide.
  • Embodiment 47 The nanovesicle drug carrier of embodiment 46, wherein said drug carrier is conjugated to a peptide that binds a receptor on a cancer cell or tumor blood vessel.
  • Embodiment 48 The nanovesicle drug carrier of embodiment 47, wherein said drug carrier is conjugated to an iRGD peptide.
  • Embodiment 49 The nanovesicle drug carrier of embodiment 47, wherein said drug carrier is conjugated to a targeting peptide shown in Table 5.
  • Embodiment 50 The nanovesicle drug carrier according to any one of embodiments 46-49, wherein said drug carrier is conjugated to transferrin, and/or ApoE, and/or folate.
  • Embodiment 51 The nanovesicle drug carrier according to any one of embodiments 46-50, wherein said drug carrier is conjugated to a targeting moiety that comprises an antibody that binds to a cancer marker.
  • Embodiment 52 The nanovesicle drug carrier of embodiment 51, wherein said drug carrier is conjugated to a targeting moiety that comprises an antibody that binds a cancer marker shown in Table 4.
  • Embodiment 53 The nanovesicle drug carrier according to any one of embodiments 51-52, wherein said antibody comprises a full-length antibody (intact immunoglobulin).
  • Embodiment 54 The nanovesicle drug carrier according to any one of embodiments 51-52, wherein said antibody comprises an antibody fragment.
  • Embodiment 55 The nanovesicle drug carrier according to any one of embodiments 51-52, wherein said antibody is selected from the group consisting of an intact immunoglobulin, an F(ab)'2, a Fab, a single chain antibody, a diabody, an affibody, a unibody, and a nanobody.
  • Embodiment 56 The nanovesicle drug carrier according to any one of embodiments 1-55, wherein said drug carriers in suspension are stable for at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months when stored at 4°C.
  • Embodiment 57 The nanovesicle drug carrier according to any one of embodiments 1-56, wherein said nanoparticle drug carrier forms a stable suspension on rehydration after lyophilization.
  • Embodiment 58 The nanovesicle drug carrier according to any one of embodiments 1-57, wherein said nanoparticle drug carriers, show reduced drug toxicity as compared to free drug.
  • Embodiment 59 The nanovesicle drug carrier according to any one of embodiments 1-58, wherein said nanoparticle drug carrier has colloidal stability in physiological fluids with pH 7.4 and remains monodisperse to allow systemic biodistribution and is capable of entering a disease site by vascular leakage (EPR effect) or transcytosis.
  • EPR effect vascular leakage
  • Embodiment 60 The nanovesicle drug carrier according to any one of embodiments 1-59, wherein said cargo within said vesicle comprises an agent that induces immunogenic cell death (ICD) selected from the group consisting of oxaliplatin,
  • ICD immunogenic cell death
  • anthracenedione bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, cyclophosphamide, and a bioreactive nanomaterial that induces ICD.
  • Embodiment 61 The nanovesicle drug carrier of embodiment 60, wherein said cargo comprises oxaliplatin.
  • Embodiment 62 The nanovesicle drug carrier of embodiment 60, wherein said cargo comprises doxorubicin.
  • Embodiment 63 The nanovesicle drug carrier of embodiment 60, wherein said cargo comprises a bioreactive nanomaterial that induces ICD and/or innate immune activation.
  • Embodiment 64 The nanovesicle drug carrier of embodiment 63, wherein said cargo comprises a nanomaterial that induces ICD where said nanomaterial is selected from the group consisting of CuO, Cu 2 0, Sb 2 0 3 , As 2 0 3 , Bi 2 0 3 , P 2 0 3 , ZnO, Ti0 2 , graphene oxide, and bioreactive 2D materials other than graphene or graphene oxide.
  • Embodiment 65 The nanovesicle drug carrier of embodiment 64, wherein said nanomaterial comprises copper oxide (e.g. CuO).
  • Embodiment 66 The nanovesicle drug carrier of embodiment 64, wherein said nanomaterial comprises Sb 2 0 3 .
  • Embodiment 67 A method of treating a cancer, said method comprising:
  • Embodiment 68 The method of embodiment 67, wherein the ICD inducer and the IDO inhibitor are synergistic in their activity against said cancer.
  • Embodiment 69 The method according to any one of embodiments 67-68, wherein said ICD-inducer is in an amount effective to elevate calreticulin (CRT) expression in cells of said cancer.
  • CRT calreticulin
  • Embodiment 70 The method according to any one of embodiments 67-69, wherein said said ICD-inducer is in an amount effective to elevate expression and/or release of HMGB 1 and/or induction of ATP release.
  • Embodiment 71 The method according to any one of embodiments 67-70, wherein said administering to a subject in need thereof an effective amount of a nanovesicle drug carrier comprises a primary therapy in a chemotherapeutic regimen.
  • Embodiment 72 The method according to any one of embodiments 67-70, wherein said administering to a subject in need thereof an effective amount of a nanovesicle drug carrier comprises an adjunct therapy in a treatment regime that additionally comprises chemotherapy using another chemotherapeutic agent, and/or surgical resection of a tumor mass, and/or radiotherapy.
  • Embodiment 73 The method according to any one of embodiments 67-72, wherein said nanoparticle drug carrier and/or said pharmaceutical formulation is a component in a multi-drug chemotherapeutic regimen.
  • Embodiment 74 The method according to any one of embodiments 67-73, wherein said cancer is pancreatic ductal adenocarcinoma (PDAC).
  • Embodiment 75 The method according to any one of embodiments 67-73, wherein said cancer is a cancer selected from the group consisting of breast cancer, lung cancer, melanoma, pancreas cancer, liver cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi sarcoma, lymphoma), anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma,
  • ALL acute lymphoblastic
  • myeloproliferative disorders colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer,
  • duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer
  • esthesioneuroblastoma extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kaposi
  • myeloma/plasma cell neoplasm mycosis fungoides, myelodysplastic syndromes,
  • Embodiment 76 The method according to any one of embodiments 67-75, wherein said administration is via a route selected from the group consisting of intravenous administration, intraarterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation (including intranasal and intratracheal delivery, intracranial administration via a cannula, and subcutaneous or intramuscular depot deposition.
  • Embodiment 77 The method according to any one of embodiments 67-75, wherein said administration comprises systemic administration via injection or cannula.
  • Embodiment 78 The method according to any one of embodiments 67-75, wherein said administration is administration to an intra-tumoral or peri-tumoral site.
  • Embodiment 79 The method according to any one of embodiments 67-78, wherein said mammal is a human.
  • Embodiment 80 The method according to any one of embodiments 67-78, wherein said mammal is a non-human mammal.
  • Embodiment 81 The method according to any one of embodiments 67-80, wherein said cancer comprises PDAC, and said IDO pathway inhibitor comprises an agent selected from the group consisting of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l- methyl-tryptophan (L-1MT), and a mixture of D-1MT and L-1MT, epacadostat.
  • said cancer comprises PDAC
  • said IDO pathway inhibitor comprises an agent selected from the group consisting of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l- methyl-tryptophan (L-1MT), and a mixture of D-1MT and L-1MT, epacadostat.
  • Embodiment 82 The method of embodiment 81, wherein said inhibitor of an
  • IDO pathway comprises D-l -methyl-try ptophan (indoximod, D-1MT).
  • Embodiment 83 The method of embodiment 81, wherein said inhibitor of an
  • Embodiment 84 The method of embodiment 81, wherein said inhibitor of an
  • IDO pathway comprises a mixture of D-1MT and L-1MT.
  • Embodiment 85 The method according to any one of embodiments 67-84, wherein said cargo within said vesicle comprises one or more drugs selected from the group consisting of doxorubicin, paclitaxel, docetaxel, cyclophosphamide, mitroxantrone, etoposide, and bortezomib.
  • said cargo within said vesicle comprises one or more drugs selected from the group consisting of doxorubicin, paclitaxel, docetaxel, cyclophosphamide, mitroxantrone, etoposide, and bortezomib.
  • Embodiment 86 The method of embodiment 85, wherein said cargo within said vesicle comprises doxorubicin.
  • Embodiment 87 The method according to any one of embodiments 85-86, wherein said cancer is a breast cancer.
  • Embodiment 88 The method according to any one of embodiments 67-84, wherein said cargo within said vesicle comprises one or more drugs selected from the group consisting of oxaliplatin, gemcitabine, taxanes (e.g., paclitaxel and docetaxel), doxorubicin, and etoposide.
  • Embodiment 89 The method of embodiment 88, wherein said cargo within said vesicle comprises oxaliplatin.
  • Embodiment 90 The method according to any one of embodiments 88-89, wherein said cancer comprise pancreatic cancer.
  • Embodiment 91 The method according to any one of embodiments 67-84, wherein said cargo within said vesicle comprises one or more drugs selected from the group consisting of a taxane (e.g., paclitaxel and docetaxel), gemcitabine, a Pt-based drug (e.g., cisplatin, oxaliplatin, carboplatin), cyclophosphamide, oxaliplatin plus cyclophosphamide, epirubicin (anthracycline), and etoposide.
  • a taxane e.g., paclitaxel and docetaxel
  • gemcitabine e.g., a Pt-based drug (e.g., cisplatin, oxaliplatin, carboplatin), cyclophosphamide, oxaliplatin plus cyclophosphamide, epirubicin (anthracycline), and etoposide.
  • Embodiment 92 The method of embodiment 91, wherein said cancer comprises lung cancer.
  • Embodiment 93 The method according to any one of embodiments 67-92, wherein said nanovesicle drug carrier is administered in conjunction with administration of an immune checkpoint inhibitor.
  • Embodiment 94 The method of embodiment 93, wherein said immune checkpoint inhibitor comprises an inhibitor of PD-1, PD-L1, PD-L2, PD-L3, PD-L4, CTLA- 4, LAG3, B7-H3, B7-H4, KIR and/or TIM3.
  • Embodiment 95 The method of embodiment 94, wherein said checkpoint inhibitor comprises an antibody that inhibits a moiety selected from the group consisting of PD-1, PD-L1, and CTLA4.
  • Embodiment 96 The method of embodiment 95, wherein said antibody comprises an antibody that inhibits PD-1.
  • Embodiment 97 The method of embodiment 96, wherein said antibody comprises Pembrolizumab (Keytruda), or Nivolumab (Opdivo).
  • Embodiment 98 The method of embodiment 95, wherein said antibody comprises an antibody that inhibits PD-L1.
  • Embodiment 99 The method of embodiment 98, wherein said antibody comprises Atezolizumab (Tecentriq), Avelumab (Bavencio), or Durvalumab (Imfinzi).
  • Embodiment 100 The method of embodiment 95, wherein said antibody comprises an antibody that inhibits CTLA-4.
  • Embodiment 101 The method of embodiment 100, wherein said antibody comprises Ipilimumab (Yervoy).
  • Embodiment 102 The method according to any one of embodiments 93-101, wherein the activity of said nanovesicle drug carrier and said immune checkpoint inhibitor is synergistic.
  • Embodiment 103 A composition comprising an IDO pathway inhibitor conjugated to a moiety that forms a nanovesicle in aqueous solution.
  • Embodiment 104 The composition of embodiment 103, wherein said IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid.
  • Embodiment 105 The composition according to any one of embodiments 103-104, wherein IDO pathway inhibitor is conjugated directly to said moiety.
  • Embodiment 106 The composition according to any one of embodiments
  • IDO pathway inhibitor is conjugated to said moiety via a linker.
  • Embodiment 107 The composition of embodiment 106, wherein said linker comprises squalene.
  • Embodiment 108 The composition according to any one of embodiments
  • IDO pathway inhibitor is conjugated to PGHP.
  • Embodiment 109 The composition according to any one of embodiments
  • Embodiment 110 The composition according to any one of embodiments
  • IDO pathway inhibitor is conjugated to cholesterol (CHOL).
  • Embodiment 111 The composition according to any one of embodiments
  • IDO pathway inhibitor is conjugated to a fatty acid.
  • Embodiment 112 The composition of embodiment 111, wherein said IDO pathway inhibitor is conjugated to oleic acid or docosahexaenoic acid.
  • Embodiment 114 The composition according to any one of embodiments 104-107, wherein said IDO pathway inhibitor is conjugated to a lipid.
  • Embodiment 115 The composition of embodiment 114, wherein said IDO pathway inhibitor is conjugated to a phospholipid.
  • Embodiment 116 The composition of embodiment 115, wherein said IDO pathway inhibitor is conjugated to a phospholipid comprising a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains.
  • Embodiment 117 The composition of embodiment 116, wherein said phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC),
  • DPPC phosphatidylcholine
  • DMPC dimyristoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • DAPC diactylphosphatidylcholine
  • Embodiment 118 The composition of embodiment 116, wherein said phospholipid comprises a natural lipid selected from the group consisting of egg
  • egg PC phosphatidylcholine
  • soy phosphatidylcholine soy phosphatidylcholine
  • Embodiment 119 The composition of embodiment 116, wherein said phospholipid comprises an unsaturated fatty acid selected from the group consisting of 1,2- dimyristoleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine, l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dieicosenoyl-sn- glycero-3 -phosphocholine.
  • an unsaturated fatty acid selected from the group consisting of 1,2- dimyristoleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine, l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dieicosenoyl-sn- glycero-3 -
  • Embodiment 120 The composition of embodiment 1 16, wherein said phospholipid comprises l-palmitoy l -2-hydroxy-sn-glycero-3 -phosphocholine.
  • Embodiment 121 The composition according to any one of embodiments
  • said IDO pathway inhibitor comprises an agent selected from the group consisting of D-l-methyl-tryptophan (indoximod, D-1MT), L-l-methyl-tryptophan (L-1MT), a mixture of D-1MT and L-1MT, 1 -methyl-L-tryptophan (L- 1MT), methylthiohydantoin-dl- tryptophan (MTH-Trp, Necrostatin), ⁇ -carbolines (e.g., 3-butyl-P-carboline),
  • Naphthoquinone-based e.g., annulin-B
  • S-allyl-brassinin S-benzyl-brassinin
  • N-[2-(Indol- 3-yl)ethyl]-S-methyl-dithiocarbamate N-[2-(benzo[b]thiophen-3-yl)ethyl]-S-methyl- dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate
  • S-hexyl-brassinin N- [2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S[(naphth-2- yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]
  • Phenylimidazole-based IDO inhibitors e.g., 4-phenylimidazole
  • Exiguamine A imidodicarbonimidic diamide,N-methyl-N'-9-phenanthrenyl-monohydrochloride
  • NSC401366 l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDO l -derived peptide, NLG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole.
  • Embodiment 122 The composition according to any one of embodiments
  • Embodiment 123 The composition of embodiment 122, wherein said IDO pathway inhibitor comprises a D isomer of 1-methyl-tiyptophpan.
  • Embodiment 124 The composition of embodiment 122, wherein said IDO pathway inhibitor comprises an L isomer of 1-methyl-tiyptophpan.
  • Embodiment 125 The composition of embodiment 122, wherein said IDO pathway inhibitor comprises a mixture of D and L isomers of 1-methyl-tiyptophpan.
  • Embodiment 126 The composition of embodiment 122, wherein the lipid conjugated IDO pathway inhibitor comprises l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine.
  • Embodiment 127 The composition of embodiment 126, wherein the lipid conju ated IDO pathway inhibitor comprises a compound having the structure:
  • Embodiment 128 The composition of embodiment 127, wherein the 1- methyl-tryptophan component of said conjugated IDO pathway inhibitor comprises a "D" isomer of 1 meth l-tyrptophan (indoximod) characterized by the formula:
  • Embodiment 129 The composition of embodiment 127, wherein the 1- methyl-tryptophan component of said conjugated IDO pathway inhibitor comprises an "L" isomer of 1 methyl-tyrptophan (L-1MT) characterized by the formula:
  • Embodiment 130 The composition of embodiment 127, wherein the 1- methyl-tryptophan component of said conjugated IDO pathway inhibitor comprises a mixture of "D" and "L” isomers of 1 -methyl -tryptophan.
  • Embodiment 131 The composition according to any one of embodiments
  • lipid-conjugated IDO pathway inhibitor forms a component of a vesicle.
  • Embodiment 132 A nanoparticle drug carrier for the combined delivery of an IDO pathway inhibitor and an inducer of immunogenic cell death (ICD), said nanoparticle drug carrier comprising:
  • a mesoporous silica nanoparticle having a surface and defining a plurality of pores that are suitable to receive molecules therein;
  • a lipid bilayer coating the surface e.g., encapsulating the nanoparticle and sealing the plurality of pores
  • a first cargo comprising an inhibitor of the indoleamine 2,3- dioxygenase (IDO) pathway (IDO pathway inhibitor);
  • a second cargo comprising an agent that induces immunogenic cell death (ICD) (ICD-inducer);
  • the lipid bilayer is substantially continuous and encapsulates said nanoparticle stably sealing the plurality of pores.
  • Embodiment 133 The nanoparticle drug carrier of embodiment 132, wherein said nanoparticle drug carrier contains a predefined ratio of IDO pathway inhibitor to ICD- inducer.
  • Embodiment 134 The nanoparticle drug carrier according to any one of embodiments 132-133, wherein the IDO pathway inhibitor and the ICD inducer are synergistic in their activity against a cancer.
  • Embodiment 135 The nanoparticle drug carrier according to any one of embodiments 132-134, wherein said drug carrier, when administered systemically, delivers an amount of an ICD inducer effective to induce or to facilitate induction of immunogenic cell death of a cancer cell.
  • Embodiment 136 The nanoparticle drug carrier according to any one of embodiments 132-135, wherein said drug carrier, when administered systemically, delivers an amount of IDO pathway inhibitor to partially or fully inhibit the IDO pathway at a cancer site.
  • Embodiment 137 The nanoparticle drug carrier according to any one of embodiments 132-136, wherein said IDO pathway inhibitor, is disposed in said lipid bilayer and/or conjugated to a lipid comprising said lipid bilayer.
  • Embodiment 138 The nanoparticle drug carrier according to any one of embodiments 132-137, wherein said ICD inducer is disposed in said plurality of pores.
  • Embodiment 139 The nanoparticle drug carrier according to any one of embodiments 132-138, wherein said ICD inducer comprises a chemotherapeutic agent selected from the group consisting of oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
  • a chemotherapeutic agent selected from the group consisting of oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
  • Embodiment 140 The nanoparticle drug carrier of embodiment 139, wherein said ICD inducer comprises oxaliplatin.
  • Embodiment 141 The nanoparticle drug carrier according to any one of embodiments 132-140, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l -methyl - tryptophan (L-1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tiyptophan (L-1MT), methylthiohydantoin-dl -tryptophan (MTH-Trp, Necrostatin), ⁇ -carbolines (e.g., 3-butyl-P- carboline), Naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thi
  • Embodiment 142 The nanoparticle drug carrier of embodiment 141, wherein said IDO pathway inhibitor comprises 1 -methyl -tryptophan.
  • Embodiment 143 The nanoparticle drug carrier of embodiment 142, wherein said IDO pathway inhibitor comprises D-l-methyl-tiyptophan (indoximod).
  • Embodiment 144 The nanoparticle drug carrier of embodiment 142, wherein said IDO pathway inhibitor comprises L-l-methyl-tiyptophan (L-MT).
  • Embodiment 145 The nanoparticle drug carrier of embodiment 142, wherein said IDO pathway inhibitor comprises a mixture of L-l-methyl-tiyptophan (L-MT) and D-l- methy 1 -tryptophan .
  • Embodiment 146 The nanoparticle drug carrier according to any one of embodiments 132-145, wherein said IDO pathway inhibitor is conjugated to a component of said lipid bilayer.
  • Embodiment 147 The nanoparticle drug carrier according to any one of embodiments 132-146, wherein said lipid bilayer comprises a phospholipid.
  • Embodiment 148 The nanoparticle drug carrier of embodiment 147, wherein said lipid bilayer comprises a phospholipid, cholesterol (CHOL), an mPEG phospholipid and/or an IDO pathway inhibitor lipid conjugate.
  • said lipid bilayer comprises a phospholipid, cholesterol (CHOL), an mPEG phospholipid and/or an IDO pathway inhibitor lipid conjugate.
  • Embodiment 149 The nanoparticle drug carrier according to any one of embodiments 147-148, wherein said phospholipid comprises a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains.
  • Embodiment 150 The nanoparticle drug carrier of embodiment 149, wherein said phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC),
  • DSPC distearoylphosphatidylcholine
  • DAPC diactylphosphatidylcholine
  • Embodiment 151 The nanoparticle drug carrier of embodiment 149, wherein said phospholipid comprises a natural lipid selected from the group consisting of egg phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC).
  • egg PC egg phosphatidylcholine
  • soy phosphatidylcholine soy phosphatidylcholine
  • Embodiment 152 The nanoparticle drug carrier of embodiment 149, wherein said phospholipid comprises an unsaturated fatty acid selected from the group consisting of l,2-dimyristoleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine,l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dieicosenoyl-sn- glycero-3 -phosphocholine.
  • DOPC 1,2-dieicosenoyl-sn- glycero-3 -phosphocholine.
  • Embodiment 153 The nanoparticle drug carrier according to any one of embodiments 147-152, wherein said lipid bilayer comprises an mPEG phospholipid with a phospholipid C14-C18 carbon chain, and a PEG molecular weight ranging from about 350 Da to 5000 Da.
  • Embodiment 154 The nanoparticle drug carrier of embodiment 153, wherein said lipid bilayer comprises l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (DSPE- PEG).
  • Embodiment 155 The nanoparticle drug carrier of embodiment 154, wherein said lipid bilayer comprises DPSE-PEG 2K .
  • Embodiment 156 The nanoparticle drug carrier according to any one of embodiments 147-155, wherein said IDO pathway inhibitor is conjugated to a moiety that forms a component of a lipid bilayer comprising a nanovesicle in aqueous solution and is provided in said lipid bilayer.
  • Embodiment 157 The nanoparticle drug carrier of embodiment 156, wherein said IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid.
  • Embodiment 158 The nanoparticle drug carrier according to any one of embodiments 156-157, wherein said IDO pathway inhibitor is conjugated directly to said moiety.
  • Embodiment 159 The nanoparticle drug carrier according to any one of embodiments 156-157, wherein said IDO pathway inhibitor is conjugated to said moiety via a linker.
  • Embodiment 160 The nanoparticle drug carrier according to any one of embodiments 156-159, wherein said IDO pathway inhibitor is conjugated to PGHP.
  • Embodiment 161 The nanoparticle drug carrier according to any one of embodiments 156-159, wherein said IDO pathway inhibitor is conjugated to vitamin E.
  • Embodiment 162 The nanoparticle drug carrier according to any one of embodiments 156-159, wherein said IDO pathway inhibitor is conjugated to cholesterol (CHOL), or squalene.
  • Embodiment 163 The nanoparticle drug carrier according to any one of embodiments 156-159, wherein said IDO pathway inhibitor is conjugated to a fatty acid.
  • Embodiment 164 The nanoparticle drug carrier of embodiment 163, wherein said i IDO pathway inhibitor is conjugated to oleic acid or docosahexaenoic acid.
  • Embodiment 166 The nanoparticle drug carrier according to any one of embodiments 156-159, wherein said IDO pathway inhibitor is conjugated to a lipid.
  • Embodiment 167 The nanoparticle drug carrier of embodiment 166, wherein said IDO pathway inhibitor is conjugated to a phospholipid comprising said lipid bilayer.
  • Embodiment 168 The nanoparticle drug carrier of embodiment 167, wherein said IDO pathway inhibitor is conjugated to l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine (PL).
  • PL l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine
  • Embodiment 169 The nanoparticle drug carrier of embodiment 168, wherein the IDO athway inhibitor conjugated to a phospholipid has the structure:
  • Embodiment 170 The nanoparticle drug carrier of embodiment 169, wherein the 1-methyl-tiyptophan component of said conjugated IDO pathway inhibitor comprises a "D han (indoximod) characterized by the formula:
  • Embodiment 171 The nanoparticle drug carrier of embodiment 169, wherein the 1-methyl-tiyptophan component of said conjugated IDO pathway inhibitor comprises an "L" isomer of 1 methyl-tyrptophan (L-1MT) characterized by the formula:
  • Embodiment 172 The nanoparticle drug carrier of embodiment 169, wherein the 1-methyl-tryptophan component of said conjugated IDO pathway inhibitor comprises a mixture of "D" and “L” isomers of 1-methyl-tryptophan.
  • Embodiment 173 The nanoparticle drug carrier according to any one of embodiments 169-172, wherein said lipid bilayer comprises IND-PL/Chol/DSPE-PEG.
  • Embodiment 174 The nanoparticle drug carrier of embodiment 173, wherein said lipid bilayer comprises about 75% IND-PL, about 20% cholesterol, and about 5% DSPE- PEG 2K .
  • Embodiment 175 The nanoparticle drug carrier according to any one of embodiments 132-174, wherein said lipid bilayer forms a substantially uniform and intact bilayer encompassing the entire nanoparticle.
  • Embodiment 176 The nanoparticle drug carrier drug carrier according to any one of embodiments 132-175, wherein said mesoporous silica nanoparticle is colloidally stable.
  • Embodiment 177 The nanoparticle drug carrier according to any one of embodiments 132-176, wherein said mesoporous silica has: an average pore size that ranges from about 1 to about 20 nm, or from about 1 to about 10 nm, or from about 2 to about 8 nm; and an average size ranging from about 50 nm up to about 300 nm, or from about 50 up to about 200 nm, or from about 50 up to about 150 nm, or from about 50 up to about 100 nm, or from about 50 up to about 80 nm, or from about 50 up to about 70 nm, or from about 60 up to about 70 nm.
  • Embodiment 178 The nanoparticle drug carrier according to any one of embodiments 132-177, wherein when the drug in the nanocarrier is a weak base, said carrier comprises a cargo-trapping agent.
  • Embodiment 179 The nanoparticle drug carrier of embodiment 178, wherein said cargo trapping agent before reaction with the drug loaded in the nanoparticle drug carrier, is selected from the group consisting of triethylammonium sucrose octasulfate (TEA 8 SOS), ( H 4 ) 2 S0 4 , an ammonium salt, a trimethylammonium salt, and a
  • Embodiment 180 The nanoparticle drug carrier of embodiment 179, wherein cargo-trapping agent before reaction with said drug is triethylammonium sucrose octasulfate (TEA 8 SOS).
  • TAA 8 SOS triethylammonium sucrose octasulfate
  • Embodiment 181 The nanoparticle drug carrier according to any one of embodiments 179-180, wherein said drug is protonated and trapped in said pores as a gel-like precipitate in association of SOS 8" .
  • Embodiment 182 The nanoparticle drug carrier according to any one of embodiments 132-181, wherein said drug carrier is conjugated to a moiety selected from the group consisting of a targeting moiety, a fusogenic peptide, and a transport peptide.
  • Embodiment 183 The nanoparticle drug carrier of embodiment 182, wherein said drug carrier is conjugated to a peptide that binds a receptor on a cancer cell or tumor blood vessel.
  • Embodiment 184 The nanoparticle drug carrier of embodiment 183, wherein said drug carrier is conjugated to an iRGD peptide.
  • Embodiment 185 The nanoparticle drug carrier of embodiment 183, wherein said drug carrier is conjugated to a targeting peptide shown in Table 5.
  • Embodiment 186 The nanoparticle drug carrier according to any one of embodiments 182-185, wherein said drug carrier is conjugated to transferrin, and/or ApoE, and/or folate.
  • Embodiment 187 The nanoparticle drug carrier according to any one of embodiments 182-186, wherein said drug carrier is conjugated to a targeting moiety that comprises an antibody that binds to a cancer marker.
  • Embodiment 188 The nanoparticle drug carrier of embodiment 187, wherein said antibody comprises a full-length antibody (intact immunoglobuloin).
  • Embodiment 189 The nanoparticle drug carrier of embodiment 187, wherein said antibody comprises an antibody fragment.
  • Embodiment 190 The nanoparticle drug carrier of embodiment 187, wherein said antibody is selected from the group consisting of an intact immunoglobulin, an F(ab)'2, a Fab, a single chain antibody, a diabody, an affibody, a unibody, and a nanobody.
  • Embodiment 191 The nanoparticle drug carrier according to any one of embodiments 187-190, wherein said antibody comprise an antibody that binds a cancer marker shown in Table 4.
  • Embodiment 192 The nanoparticle drug carrier according to any one of embodiments 132-191, wherein said drug carriers in suspension are stable for at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months when stored at 4°C.
  • Embodiment 193 The nanoparticle drug carrier according to any one of embodiments 132-192, wherein said nanoparticle drug carrier forms a stable suspension on rehydration after lyophilization.
  • Embodiment 194 The nanoparticle drug carrier according to any one of embodiments 132-193, wherein said nanoparticle drug carriers, show reduced drug toxicity as compared to free drug and/or drug in a liposome.
  • Embodiment 195 The nanoparticle drug carrier according to any one of embodiments 132-194, wherein said nanoparticle drug carrier has colloidal stability in physiological fluids with pH 7.4 and remains monodisperse to allow systemic biodistribution and is capable of entering a disease site by vascular leakage (EPR effect) or transcytosis.
  • EPR effect vascular leakage
  • Embodiment 196 A nanomaterial carrier for the combined delivery of an inhibitor of the IDO pathway and an inducer of immunogenic cell death (ICD), said nanomaterial carrier comprising:
  • lipid or lipid formulation comprising an IDO pathway inhibitor where said lipid or lipid formulation is disposed on the surface of said nanomaterial.
  • Embodiment 197 The nanomaterial carrier of embodiment 196, wherein said lipid or lipid formulation fully encapsulates said nanomaterial.
  • Embodiment 198 The nanomaterial carrier according to any one of embodiments 196-197, wherein said lipid or lipid formulation is not a lipid bilayer.
  • Embodiment 199 The nanomaterial carrier according to any one of embodiments 196-197, wherein said lipid or lipid formulation comprises a lipid bilayer.
  • Embodiment 200 The nanomaterial carrier according to any one of embodiments 196-199, wherein said nanomaterial comprises a material selected from the group consisting of selected from the group consisting of CuO, Cu 2 0, Sb 2 0 3 , As 2 0 3 , Bi 2 0 3 , P 2 0 3 , ZnO, Ti0 2 , graphene oxide, and 2D materials other than graphene or graphene oxide.
  • Embodiment 201 The nanomaterial carrier of embodiment 200, wherein said nanomaterial comprises copper oxide (CuO).
  • Embodiment 202 The nanomaterial carrier according to any one of embodiments 200-201, wherein said nanomaterial comprises Sb 2 0 3 .
  • Embodiment 203 The nanomaterial carrier according to any one of embodiments 200-202, wherein said material comprise a 2D nanomaterial.
  • Embodiment 204 The nanomaterial carrier of embodiment 203, said 2D nanomaterial comprises a material selected from the group consisting of graphene, graphyne, borophene, germanene, silicene, Si 2 BN, stanene, phosphorene, bismuthene, molybdenite, metals, and a 2D supracrystal.
  • Embodiment 205 The nanomaterial carrier according to any one of embodiments 200-204, wherein said nanomaterial comprises graphene oxide (GO).
  • GO graphene oxide
  • Embodiment 206 The nanomaterial carrier according to any one of embodiments 196-205, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l -methyl - tryptophan (L-1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tiyptophan (L-1MT), methylthiohydantoin-dl -tryptophan (MTH-Trp, Necrostatin), ⁇ -carbolines (e.g., 3-butyl-P- carboline), Naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3
  • NSC401366 l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDOl -derived peptide, LG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole.
  • Embodiment 207 The nanomaterial carrier according to any one of embodiments 196-205, wherein said IDO pathway inhibitor comprises 1 -methyl tryptophan.
  • Embodiment 208 The nanomaterial carrier of embodiment 207, wherein said
  • IDO pathway inhibitor comprises a D isomer of 1 -methyl tryptophan.
  • Embodiment 209 The nanomaterial carrier of embodiment 207, wherein said
  • IDO pathway inhibitor comprises an L isomer of 1 -methyl tryptophan.
  • Embodiment 210 The nanomaterial carrier of embodiment 207, wherein said
  • IDO pathway inhibitor comprises a mixture of D and L isomers of 1 -methyl tryptophan.
  • Embodiment 211 The nanomaterial carrier to any one of embodiments 196-
  • Embodiment 212 The nanomaterial carrier of embodiment 211, wherein said
  • IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid.
  • Embodiment 213 The nanomaterial carrier according to any one of embodiments 211-212, wherein said IDO pathway inhibitor is conjugated directly to said moiety.
  • Embodiment 214 The nanomaterial carrier according to any one of embodiments 211-212, wherein said IDO pathway inhibitor is conjugated to said moiety via a linker.
  • Embodiment 215 The nanomaterial carrier according to any one of embodiments 212-214, wherein said IDO pathway inhibitor is conjugated to PGHP.
  • Embodiment 216 The nanomaterial carrier according to any one of embodiments 212-214, wherein said IDO pathway inhibitor is conjugated to vitamin E.
  • Embodiment 217 The nanomaterial carrier according to any one of embodiments 212-214, wherein said IDO pathway inhibitor is conjugated to cholesterol (CHOL).
  • Embodiment 218 The nanomaterial carrier according to any one of embodiments 212-214, wherein said IDO pathway inhibitor is conjugated to a fatty acid.
  • Embodiment 219 The nanomaterial carrier of embodiment 218, wherein said inhibitor of the IDO pathway is conjugated to oleic acid or docosahexaenoic acid.
  • Embodiment 220 The nanomaterial carrier of embodiment 219, wherein said
  • Embodiment 221 The nanomaterial carrier according to any one of embodiments 212-214, wherein said IDO pathway inhibitor is conjugated to a lipid.
  • Embodiment 222 The nanomaterial carrier of embodiment 221, wherein said
  • IDO pathway inhibitor is conjugated to a phospholipid.
  • Embodiment 223 The nanomaterial carrier of embodiment 222, wherein said inhibitor of the IDO pathway is conjugated to a phospholipid comprising a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains.
  • Embodiment 224 The nanomaterial carrier of embodiment 223, wherein said phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC),
  • DPPC phosphatidylcholine
  • DMPC dimyristoylphosphatidylcholine
  • Embodiment 225 The nanomaterial carrier of embodiment 223, wherein said phospholipid comprises a natural lipid selected from the group consisting of egg
  • egg PC phosphatidylcholine
  • soy phosphatidylcholine soy phosphatidylcholine
  • Embodiment 226 The nanomaterial carrier of embodiment 223, wherein said phospholipid comprises an unsaturated fatty acid selected from the group consisting of 1,2- dimyristoleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine,l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dieicosenoyl-sn- glycero-3 -phosphocholine.
  • DOPC dioleoyl-sn-glycero-3-phosphocholine
  • Embodiment 227 The nanomaterial carrier of embodiment 223, wherein said phospholipid comprises l-palmitoyl-2-hydroxy-sn-glycero-3 -phosphocholine.
  • Embodiment 228 The nanomaterial carrier according to any one of embodiments 196-227, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of of D-l-methyl-tryptophan (indoximod, D-1MT), L-l -methyl - tryptophan (L-1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tryptophan (L-1MT), methylthiohydantoin-dl -tryptophan (MTH-Trp, Necrostatin), ⁇ -carbolines (e.g., 3-butyl-P- carboline), Naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassin
  • NSC401366 l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDO l -derived peptide, NLG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole.
  • Embodiment 229 The nanomaterial carrier according to any one of embodiments 196-227, wherein said IDO pathway inhibitor comprises 1 -methyl -tryptophan.
  • Embodiment 230 The nanomaterial carrier of embodiment 229, wherein said
  • Embodiment 231 The nanomaterial carrier of embodiment 229, wherein said
  • 1 -methyl -tryptophan comprises an L isomer of 1 -methyl -tryptophan.
  • Embodiment 232 The nanomaterial carrier of embodiment 229, wherein said
  • 1 -methyl -tryptophan comprises a mixture of D and L isomers of 1 -methyl -tryptophan.
  • Embodiment 233 The nanomaterial carrier of embodiment 229, wherein said 1 -methyl -tryptophan is conjugated to l -palmitoy l -2-hydroxy-s «-glycero-3-phosphocholine.
  • Embodiment 234 The nanomaterial carrier of embodiment 229, wherein said
  • Embodiment 235 The nanomaterial carrier of embodiment 234, wherein the
  • 1 -methyl -tryptophan component of said conjugated IDO pathway inhibitor comprises a "D" isom r of 1 methyl-tyrptophan (indoximod) characterized by the formula:
  • Embodiment 236 The nanovesicle drug carrier of embodiment 234, wherein the 1-methyl-tiyptophan component of said conjugated IDO pathway inhibitor comprises an "L" isomer of 1 methyl-tyrptophan (L-1MT) characterized by the formula:
  • Embodiment 237 The nanovesicle drug carrier of embodiment 234, wherein the 1-methyl-tiyptophan component of said conjugated IDO pathway inhibitor comprises a mixture of "D" and "L” isomers of 1 -methyl -tryptophan.
  • Embodiment 238 The nanomaterial carrier according to any one of embodiments 196-237, wherein said ICD inducer and said IDO pathway inhibitor are synergistic in their activity on a cancer.
  • Embodiment 239 The nanomaterial carrier according to any one of embodiments 196-238, wherein said effective amount of said ICD-inducer is an amount effective to elevate calreticulin (CRT) expression in cancer cells at the tumor site.
  • CRT calreticulin
  • Embodiment 240 The nanomaterial carrier according to any one of embodiments 196-239, wherein said effective amount of said ICD-inducer is an amount effective to elevate expression and/or release of HMGB1 and/or induction of ATP release in cancer cells at the tumor site.
  • Embodiment 241 The nanomaterial carrier according to any one of embodiments 196-240, wherein said drug carrier is conjugated to a moiety selected from the group consisting of a targeting moiety, a fusogenic peptide, and a transport peptide.
  • Embodiment 242 The nanomaterial carrier of embodiment 241, wherein said drug carrier is conjugated to a peptide that binds a receptor on a cancer cell or tumor blood vessel at the tumor site.
  • Embodiment 243 The nanomaterial carrier of embodiment 242, wherein said drug carrier is conjugated to an iRGD peptide.
  • Embodiment 244 The nanomaterial carrier of embodiment 242, wherein said drug carrier is conjugated to a targeting peptide shown in Table 5.
  • Embodiment 245 The nanomaterial carrier according to any one of embodiments 241-244, wherein said drug carrier is conjugated to transferrin, and/or ApoE, and/or folate.
  • Embodiment 246 The nanomaterial carrier according to any one of embodiments 241-245, wherein said drug carrier is conjugated to a targeting moiety that comprises an antibody that binds to a cancer marker.
  • Embodiment 247 The nanomaterial carrier of embodiment 246, wherein said drug carrier is conjugated to a targeting moiety that comprises an antibody that binds a cancer marker shown in Table 4.
  • Embodiment 248 A pharmaceutical formulation said formulation comprising:
  • Embodiment 249 The formulation of embodiment 248, wherein said formulation comprises a plurality of nanoparticle drug carriers according to any one of embodiments 132-195.
  • Embodiment 250 The formulation of embodiment 248, wherein said formulation comprises a plurality of nanovesicle drug carriers according to any one of embodiments 1-59.
  • Embodiment 251 The formulation of embodiment 248, wherein said formulation comprises a plurality of nanomaterial carriers according to any one of embodiments 196-254.
  • Embodiment 252 The formulation according to any one of embodiments of embodiments 248-251, wherein said formulation is an emulsion, dispersion, or suspension.
  • Embodiment 253 The formulation of embodiment 252, wherein said suspension, emulsion, or dispersion is stable for at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months when stored at 4°C.
  • Embodiment 254 The formulation according to any one of embodiments
  • the nanoscale drug carriers in said formulation show a substantially unimodal size distribution; and/or the drug carriers in said suspension, emulsion, or dispersion shows a PDI less than about 0.2, or less than about 0.1.
  • Embodiment 255 The formulation according to any one of embodiments
  • said formulation is formulated for administration via a route selected from the group consisting of intravenous administration, intraarterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation (including intranasal and intratracheal delivery, intracranial administration via a cannula, and subcutaneous or intramuscular depot deposition.
  • Embodiment 256 The formulation according to any one of embodiments
  • Embodiment 257 The formulation according to any one of embodiments
  • Embodiment 258 A method of treating a cancer in a mammal, said method comprising:
  • ICD immunogenic cell death
  • Embodiment 259 The method of embodiment 258, wherein said ICD inducer and said IDO pathway inhibitor are synergistic in their activity on said cancer.
  • Embodiment 260 The method according to any one of embodiments 258-
  • said effective amount of said ICD-inducer is an amount effective to elevate calreticulin (CRT) expression in cells of said cancer at the tumor site.
  • CRT calreticulin
  • Embodiment 261 The method according to any one of embodiments 258-
  • Embodiment 262 The method according to any one of embodiments 258-
  • IDO pathway inhibitor and said ICD inducer are provided as a nanoparticle drug carrier according to any one of embodiments 132-195.
  • Embodiment 263 The method according to any one of embodiments 258-
  • nanomaterial carrier according to any one of embodiments 196-254.
  • Embodiment 264 The method according to any one of embodiments 258-
  • IDO pathway inhibitor is conjugated to a moiety that forms a component of a lipid bilayer comprising a vesicle in aqueous solution.
  • Embodiment 265 The method of embodiment 264, wherein said IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid.
  • Embodiment 266 The method according to any one of embodiments 264-
  • Embodiment 267 The method according to any one of embodiments 264- 265, wherein said IDO pathway inhibitor is conjugated to said moiety via a linker.
  • Embodiment 268 The method according to any one of embodiments 265-
  • Embodiment 269 The method according to any one of embodiments 265-
  • Embodiment 270 The method according to any one of embodiments 265-
  • Embodiment 271 The method according to any one of embodiments 265-
  • Embodiment 272 The method of embodiment 271, wherein said IDO pathway inhibitor is conjugated to oleic acid or docosahexaenoic acid.
  • Embodiment 274 The method according to any one of embodiments 265- 267, wherein said IDO pathway inhibitor is conjugated to a lipid.
  • Embodiment 275 The method of embodiment 274, wherein said IDO pathway inhibitor is conjugated to a phospholipid.
  • Embodiment 276 The method of embodiment 275, wherein said IDO pathway inhibitor is conjugated to a phospholipid comprising a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains.
  • Embodiment 277 The method of embodiment 276, wherein said
  • phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC),
  • DSPC distearoylphosphatidylcholine
  • DAPC diactylphosphatidylcholine
  • Embodiment 278 The method of embodiment 276, wherein said
  • phospholipid comprises a natural lipid selected from the group consisting of egg
  • egg PC phosphatidylcholine
  • soy phosphatidylcholine soy phosphatidylcholine
  • Embodiment 279 The method of embodiment 276, wherein said
  • phospholipid comprises an unsaturated fatty acid selected from the group consisting of 1,2- dimyristoleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine, l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dieicosenoyl-sn- glycero-3 -phosphocholine.
  • 1,2- dimyristoleoyl-sn-glycero-3-phosphocholine 1,2- dimyristoleoyl-sn-glycero-3-phosphocholine
  • DOPC dioleoyl-sn-glycero-3-phosphocholine
  • DOPC 1,2-dieicosenoyl-sn- glycero-3 -phosphocholine
  • Embodiment 280 The method of embodiment 276, wherein said
  • phospholipid comprises l-palmitoy l -2-hydroxy-sn-glycero-3 -phosphocholine.
  • Embodiment 281 The method according to any one of embodiments 258-
  • said IDO pathway inhibitor comprises an agent selected from the group consisting of of D-l-methyl-tryptophan (indoximod, D-1MT), L-l-methyl-tryptophan (L- 1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tryptophan (L-1MT),
  • methylthiohydantoin-dl-tryptophan MTH-Trp, Necrostatin
  • ⁇ -carbolines e.g., 3-butyl-P- carboline
  • Naphthoquinone-based e.g., annulin-B
  • S-allyl-brassinin S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S- methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl- brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol-3
  • NSC401366 l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDO l -derived peptide, NLG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole.
  • Embodiment 282 The method according to any one of embodiments 258-
  • Embodiment 283 The method of embodiment 282, wherein said IDO pathway inhibitor comprises a substantially pure D enantiomer of 1-methyl-tiyptophan.
  • Embodiment 284 The method of embodiment 282, wherein said inhibitor of
  • IDO pathway inhibitor comprises a substantially pure L enantiomer of 1-methyl-tiyptophan.
  • Embodiment 285 The method of embodiment 284, wherein said 1-methyl- tryptophan is conjugated to l -palmitoy l-2-hydroxy-5 «-glycero-3 -phosphocholine.
  • Embodiment 286 The method of embodiment 285, wherein said 1-methyl- tryptophan conjugated to l -palmitoy l -2-hydroxy-5 «-glycero-3 -phosphocholine comprises a com ound having the structure:
  • Embodiment 287 The method of embodiment 286, wherein the 1-methyl- tryptophan component of said conjugated IDO pathway inhibitor comprises a "D" isomer methyl-tyrptophan (indoximod) characterized by the formula:
  • Embodiment 288 The method of embodiment 286, wherein the 1-methyl- tryptophan component of said conjugated IDO pathway inhibitor comprises an "L" isomer of 1 meth -tyrptophan (L-1MT) characterized by the formula:
  • Embodiment 289 The method of embodiment 286, wherein the 1-methyl- tryptophan component of said conjugated IDO pathway inhibitor comprises a mixture of "D" and "L” isomers of 1-methyl-tiyptophan.
  • Embodiment 290 The method according to any one of embodiments 274-
  • lipid-conjugated IDO pathway inhibitor forms a component of a vesicle.
  • Embodiment 291 The method according to any one of embodiments 258-
  • said agent that induces immunogenic cell death comprises an agent selected from the group consisting of oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
  • Embodiment 292 The method of embodiment 291, wherein said agent that induces immunogenic cell death (ICD) comprises oxaliplatin.
  • Embodiment 293 The method according to any one of embodiments 258-
  • said agent that induces immunogenic cell death (ICD) comprises or contains a nanomaterial that induces ICD.
  • Embodiment 294 The method of embodiment 293, wherein said
  • Embodiment 295 The method of embodiment 293, wherein said
  • nanomaterial comprises a core-shell nanoparticle.
  • Embodiment 296 The method of embodiment 293, wherein said
  • nanomaterial comprises a doped nanoparticle.
  • Embodiment 297 The method according to any one of embodiments 292- 296, wherein said agent that induces immunogenic cell death (ICD) comprises a nanomaterial selected from the group consisting of CuO, Cu 2 0, Sb 2 0 3 , As 2 0 3 , Bi 2 0 3 , P 2 0 3 , ZnO, Ti0 2 , graphene oxide, and 2D materials other than graphene or graphene oxide.
  • ICD immunogenic cell death
  • Embodiment 298 The method of embodiment 297, wherein said
  • Embodiment 299 The method of embodiment 297, wherein said
  • nanomaterial comprises Sb 2 0 3 .
  • Embodiment 300 The method of embodiment 297, wherein said material comprise a 2D nanomaterial.
  • Embodiment 301 The method of embodiment 300, said 2D nanomaterial comprise a material selected from the group consisting of graphene, graphyne, borophene, germanene, silicene, si2bn, stanene, phosphorene, bismuthene, molybdenite, metals, and a 2D supracrystal.
  • Embodiment 302 The method of embodiment 301, wherein said
  • Embodiment 303 The method according to any one of embodiments 258-
  • agent that induces immunogenic cell death comprises an oncolytic virus.
  • Embodiment 304 The method of embodiment 303, wherein said oncolytic virus comprises a virus selected from the group consisting of Parvovirus (e.g., H-PV), Adenovirus (e.g. , hTERT-Ad, Ad5/3-D24-GMCSF), Herpes simplex virus (e.g., G207, HSV- 1716, T-VEC, HSV-2 ⁇ mutant), Poxvirus (e.g., vSP, vvDDPexa-Vec), Arbovirus (e.g., VSV-GFP Indiana serotype, VSVgm-icv), and Paramyxovirus (e.g., MV-eGFP (Edmonston strain)).
  • Parvovirus e.g., H-PV
  • Adenovirus e.g. , hTERT-Ad, Ad5/3-D24-GMCSF
  • Herpes simplex virus e.g., G207, HSV- 17
  • Embodiment 305 The method according to any one of embodiments 258- 304, wherein said IDO pathway inhibitor is administered before said agent that induces immunogenic cell death (ICD) (ICD-inducer).
  • ICD immunogenic cell death
  • Embodiment 306 The method according to any one of embodiments 258-
  • Embodiment 307 The method according to any one of embodiments 258-
  • IDO pathway inhibitor inhibitor is administered simultaneously with said agent that induces immunogenic cell death (ICD) (ICD-inducer).
  • ICD immunogenic cell death
  • Embodiment 308 The method of embodiment 307, wherein said IDO pathway inhibitor and said agent that induces immunogenic cell death (ICD) (ICD-inducer) are administered as a combined formulation.
  • ICD immunogenic cell death
  • Embodiment 309 The method according to any one of embodiments 258-
  • ICD immunogenic cell death
  • Embodiment 310 The method according to any one of embodiments 258- 309, wherein said IDO pathway inhibitor and/or said agent that induces immunogenic cell death (ICD) (ICD-inducer) is delivered into a peri-tumoral site.
  • ICD immunogenic cell death
  • Embodiment 31 1 The method according to any one of embodiments 258-
  • Embodiment 312 The method according to any one of embodiments 258-
  • ICD immunogenic cell death
  • Embodiment 313 The method according to any one of embodiments 258-
  • said cancer is selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Adrenocortical carcinoma, Kaposi sarcoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma,
  • PDAC pancreatic ductal adenocarcinoma
  • ALL acute lymph
  • ependymoma breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g.
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • colon cancer colorectal cancer
  • craniopharyngioma cutaneous t-cell lymphoma
  • duct cancers e.g.
  • bile extrahepatic
  • ductal carcinoma in situ DCIS
  • embryonal tumors endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhan
  • Embodiment 314 The method according to any one of embodiments 258- 312, wherein said cancer is pancreatic ductal adenocarcinoma (PDAC).
  • PDAC pancreatic ductal adenocarcinoma
  • Embodiment 315 The method according to any one of embodiments 258-
  • Embodiment 316 The method according to any one of embodiments 258-
  • Embodiment 317 A method of treating a cancer, said method comprising: administering to a subject in need thereof an effective amount of a nanoparticle drug carrier according to any one of embodiments 132-195; and/or a nanomaterial carrier according to any one of embodiments 196-254.
  • Embodiment 318 The method of embodiment 317, wherein said method comprises administering an effective amount of a nanoparticle drug carrier according to any one of embodiments 132-195.
  • Embodiment 319 The method of embodiment 317, wherein said method comprises administering an effective amount of ICD-inducing nanomaterial carriers according to any one of embodiments 196-254.
  • Embodiment 320 The method according to any one of embodiments 317- 319, wherein the ICD inducer and the IDO pathway inhibitor are synergistic in their activity against said cancer.
  • Embodiment 321 The method according to any one of embodiments 317-
  • Embodiment 322 The method according to any one of embodiments 317-
  • Embodiment 323 The method according to any one of embodiments 317-
  • nanoparticle drug carrier and/or said pharmaceutical formulation is a component in a multi-drug chemotherapeutic regimen.
  • Embodiment 324 The method according to any one of embodiments 317-
  • Embodiment 325 The method according to any one of embodiments 317-
  • said cancer is a cancer selected from the group consisting of acute
  • lymphoblastic leukemia ALL
  • AML acute myeloid leukemia
  • Adrenocortical carcinoma AIDS-related cancers (e.g., Kaposi sarcoma, lymphoma), anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.
  • bile extrahepatic
  • ductal carcinoma in situ DCIS
  • embryonal tumors endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer ⁇ e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors ⁇ e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, lange
  • histiocytoma of bone and osteosarcoma melanoma ⁇ e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple
  • myeloma/plasma cell neoplasm mycosis fungoides, myelodysplastic syndromes,
  • Embodiment 326 The method according to any one of embodiments 317- 325, wherein said nanoparticle drug carrier is not conjugated to an iRGD peptide and the nanoparticle drug carrier is administered in conjunction with an iRGD peptide.
  • Embodiment 327 The method according to any one of embodiments 317-
  • Embodiment 328 The method of embodiment 327, wherein said immune checkpoint inhibitor comprises an inhibitor of PD-1, PD-Ll, PD-L2, PD-L3, PD-L4, CTLA- 4, LAG3, B7-H3, B7-H4, KIR and/or TIM3.
  • Embodiment 329 The method of embodiment 328, wherein said checkpoint inhibitor comrpies an antibody that inhibits a moiety selected from the group consisting of PD-1, PD-Ll, and CTLA4.
  • Embodiment 330 The method of embodiment 329, wherein said antibody comprises an antibody that inhibits PD-1.
  • Embodiment 331 The method of embodiment 330, wherein said antibody comprises Pembrolizumab (Keytruda), or Nivolumab (Opdivo).
  • Embodiment 332 The method of embodiment 329, wherein said antibody comprises an antibody that inhibits PD-Ll .
  • Embodiment 333 The method of embodiment 332, wherein said antibody comprises Atezolizumab (Tecentriq), Avelumab (Bavencio), or Durvalumab (Imfinzi).
  • Atezolizumab Tecentriq
  • Avelumab Avelumab
  • Durvalumab Imfinzi
  • Embodiment 334 The method of embodiment 329, wherein said antibody comprises an antibody that inhibits CTLA-4.
  • Embodiment 335 The method of embodiment 334, wherein said antibody comprises Ipilimumab (Yervoy).
  • Embodiment 336 The method according to any one of embodiments 327-
  • Embodiment 335 A method for the treatment and/or prevention of a cancer in a mammal, said method comprising: providing cancer cells in which immunogenic cell death (ICD) has been induced ex vivo; and vaccinating said mammal with said cells, where said vaccination induces an anti-cancer immunogenic response.
  • ICD immunogenic cell death
  • immunogenic cell death is induced by contacting said cancer cells with a chemotherapeutic agent that induces immunogenic cell death.
  • Embodiment 339 The method of embodiment 338, wherein said
  • immunogenic cell death is induced by contacting said cells with a chemotherapeutic agent selected from the group consisting of oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
  • a chemotherapeutic agent selected from the group consisting of oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
  • Embodiment 340 The method according to any one of embodiments 337-
  • immunogenic cell death is induced by contacting said cells with oxaliplatin.
  • Embodiment 341 The method according to any one of embodiments 337-
  • immunogenic cell death is induced by contacting said cells with doxorubicin.
  • Embodiment 342 The method according to any one of embodiments 337- 340, wherein said immunogenic cell death (ICD) is induced by contacting said cells with a bioreactive nanomaterial that induces ICD.
  • ICD immunogenic cell death
  • Embodiment 343 The method of embodiment 342, wherein said
  • Embodiment 344 The method according to any one of embodiments 342-
  • said ICD-inducing nanomaterial comprises a material selected from the group consisting of CuO, Cu 2 0, Sb 2 0 3 , As 2 0 3 , Bi 2 0 3 , P 2 0 3 , ZnO, Ti0 2 , graphene oxide, and 2D materials other than graphene or graphene oxide.
  • Embodiment 345 The method of embodiment 344, wherein said
  • Embodiment 346 The method according to any one of embodiments 344-
  • nanomaterial comprises Sb 2 C>3.
  • Embodiment 347 The method of embodiment 344, wherein said material comprise a 2D nanomaterial.
  • Embodiment 348 The method of embodiment 347, said 2D nanomaterial comprise a material selected from the group consisting of graphene, graphyne, borophene, germanene, silicene, si2bn, stanene, phosphorene, bismuthene, molybdenite, metals, and a 2D supracrystal.
  • Embodiment 349 The method of embodiment 348, wherein said
  • nanomaterial comprises graphene oxide (GO).
  • Embodiment 350 The method according to any one of embodiments 342-
  • nanomaterial comprises a nanoparticle.
  • Embodiment 351 The method of embodiment 348, wherein said
  • Embodiment 352 The method of embodiment 348, wherein said
  • nanomaterial comprises a doped nanoparticle.
  • Embodiment 353 The method according to any one of embodiments 337-
  • Embodiment 354 The method according to any one of embodiments 337-
  • Embodiment 355 The method of embodiment 354, wherein said oncolytic virus is selected from the group consisting of Parvovirus (e.g., H-PV), Adenovirus (e.g., hTERT-Ad, Ad5/3-D24-GMCSF), Herpes simplex virus (e.g., G207, HSV-1716, T-VEC, HSV-2 ⁇ mutant), Poxvirus (e.g., vSP, vvDDPexa-Vec), Arbovirus (e.g., VSV-GFP Indiana serotype, VSVgm-icv), and Paramyxovirus (e.g., MV-eGFP (Edmonston strain)).
  • Parvovirus e.g., H-PV
  • Adenovirus e.g., hTERT-Ad, Ad5/3-D24-GMCSF
  • Herpes simplex virus e.g., G207, HSV-1716, T-VEC, H
  • Embodiment 356 The method according to any one of embodiments 337-
  • Embodiment 357 The method according to any one of embodiments 337-
  • Embodiment 358 The method according to any one of embodiments 337-
  • cancer cells in which immunogenic cell death (ICD) is induced are of the same type of cancer that is to be treated or prevented.
  • Embodiment 359 The method according to any one of embodiments 337-
  • cancer to be treated or prevented is a cancer selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), acute lymphoblastic leukemia
  • PDAC pancreatic ductal adenocarcinoma
  • ALL acute myeloid leukemia
  • AML acute myeloid leukemia
  • adrenocortical carcinoma Kaposi sarcoma
  • anal cancer appendix cancer
  • astrocytoma atypical teratoid/rhabdoid tumor
  • bile duct cancer extrahepatic cancer
  • bladder cancer bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma)
  • brain stem glioma e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chord
  • bile extrahepatic
  • ductal carcinoma in situ DCIS
  • embryonal tumors endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhan
  • lymphoblastic ALL
  • AML acute myeloid
  • CLL chronic lymphocytic
  • CML chronic myelogenous
  • hairy cell lip and oral cavity cancer
  • liver cancer primary
  • lobular carcinoma in situ LCIS
  • lung cancer ⁇ e.g., childhood, non-small cell, small cell
  • lymphoma ⁇ e.g., AIDS-related, Burkitt ⁇ e.g., non-Hodgkin lymphoma
  • cutaneous T-Cell ⁇ e.g., mycosis fungoides, Sezary syndrome
  • Hodgkin non-Hodgkin
  • primary central nervous system CNS
  • macroglobulinemia Waldenstrom
  • male breast cancer malignant fibrous
  • histiocytoma of bone and osteosarcoma melanoma ⁇ e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple
  • myeloma/plasma cell neoplasm mycosis fungoides, myelodysplastic syndromes,
  • Embodiment 360 The method according to any one of embodiments 337-
  • cancer to be treated or prevented is selected from the group consisting of pancreatic cancer, lung cancer, breast cancer, and colon cancer.
  • Embodiment 361 The method according to any one of embodiments 337-
  • the cancer to be treated or prevented is pancreatic ductal adenocarcinoma (PDAC).
  • PDAC pancreatic ductal adenocarcinoma
  • Embodiment 362 The method according to any one of embodiments 337-
  • Embodiment 363 The method according to any one of embodiments 337-
  • cancer cells in which immunogenic cell death is induced are obtained from a primary tumor.
  • Embodiment 364 The method according to any one of embodiments 337- 363, wherein said cancer cells in which immunogenic cell death is induced are obtained from a tumor in the mammal that is to be treated.
  • Embodiment 365 The method according to any one of embodiments 337-
  • cancer cells in which immunogenic cell death is induced are obtained from a tumor biopsy, bloodstream, peritoneal fluid, pleural fluid, synovial fluid or bone marrow aspirate.
  • Embodiment 366 The method according to any one of embodiments 337-
  • cancer cells in which immunogenic cell death is induced are obtained from a resected primary or metastatic tumor.
  • Embodiment 367 The method according to any one of embodiments 337- 366, wherein said treatment is performed in the absence of surgical intervention or prior to surgical intervention.
  • Embodiment 368 The method according to any one of embodiments 337-
  • Embodiment 369 The method according to any one of embodiments 337-
  • Embodiment 370 The method according to any one of embodiments 337-
  • Embodiment 371 The method according to any one of embodiments 337-
  • Embodiment 372 The method of embodiment 371, wherein said mammal is a mammal with a family history of cancer.
  • Embodiment 373 The method according to any one of embodiments 371- 372, wherein said mammal has a genetic marker for elevated cancer risk.
  • Embodiment 374 The method according to any one of embodiments 371-
  • Embodiment 375 The method according to any one of embodiments 337-
  • cancer cells in which immunogenic cell death (ICD) is induced are from a cancer or immortalized cell line.
  • Embodiment 376 The method of embodiment 375, wherein said cancer cells in which immunogenic cell death (ICD) is induced are from a cancer cell line or
  • Embodiment 377 The method according to any one of embodiments 337- 376, wherein said vaccination comprises cutaneous and/or subcutaneous vaccination.
  • Embodiment 378 The method according to any one of embodiments 337-
  • said vaccination comprises intramuscular vaccination.
  • Embodiment 379 The method according to any one of embodiments 337-
  • Embodiment 380 The method according to any one of embodiments 337-
  • Embodiment 381 The method of embodiment 380, wherein said adjuvant is selected from the group consisting of alum, Squalene-Oil-in-water (e.g., MF59®), a PRR ligand, TLR3 and RLR Ligands, TLR4 ligands, TLR5 ligands, TLR7/8 ligands, TLR9 ligands, NOD2 ligands, and RLP3 inflammasome activators.
  • Embodiment 382 The method according to any one of embodiments 337-
  • Embodiment 383 The method according to any one of embodiments 337-
  • Embodiment 384 A kit for the treatment or prophylaxis of a cancer said kit comprising:
  • ICD-inducer an agent that induces immunogenic cell death
  • nanovesicle drug carriers according to any one of embodiments 1-59;
  • nanoparticle drug carriers according to any one of embodiments 132-195; and/or
  • nanomaterial carriers according to any one of embodiments 196-254.
  • Embodiment 385 The kit of embodiment 384, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l-methyl-tiyptophan (L-1MT), a mixture of D-1MT and L-1MT, 1- methyl-L-tryptophan (L-1MT), methylthiohydantoin-dl -tryptophan (MTH-Trp, Necrostatin), ⁇ -carbolines (e.g., 3-butyl-P-carboline), Naphthoquinone-based (e.g., annulin-B), S-allyl- brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2- (benzo[b]thiophen-3-yl)ethyl]-S
  • Embodiment 386 The kit of embodiment 384, wherein said IDO inhibitor comprises indoximod.
  • Embodiment 387 The kit of embodiment 384, wherein said IDO inhibitor comprise an IDO inhibitor conjugated to a phospholipid according to any one of
  • Embodiment 388 The kit according to any one of embodiments 384-387, wherein said ICD inducer comprises a chemotherapeutic agent selected from the group consisting of oxaliplatin, cisplatin, doxorubicin, epirubicin, idarubicin, mitoxantrone, anthracenedione, bleomycin, bortezomib, R2016, and cyclophosphamide.
  • a chemotherapeutic agent selected from the group consisting of oxaliplatin, cisplatin, doxorubicin, epirubicin, idarubicin, mitoxantrone, anthracenedione, bleomycin, bortezomib, R2016, and cyclophosphamide.
  • Embodiment 389 The kit of embodiment 388, wherein said ICD inducer comprises oxaliplatin.
  • Embodiment 390 The kit according to any one of embodiments 384-389, wherein said ICD inducer comprises an oncolytic virus.
  • Embodiment 391 The kit of embodiment 390, wherein said oncolytic virus is selected from the group consisting of Parvovirus (e.g., H-PV), Adenovirus (e.g., hTERT-Ad, Ad5/3-D24-GMCSF), Herpes simplex virus (e.g., G207, HSV-1716, T-VEC, HSV-2 ⁇ mutant), Poxvirus (e.g., vSP, vvDDPexa-Vec), Arbovirus (e.g., VSV-GFP Indiana serotype, VSVgm-icv), and Paramyxovirus (e.g., MV-eGFP (Edmonston strain)).
  • Parvovirus e.g., H-PV
  • Adenovirus e.g., hTERT-Ad, Ad5/3-D24-GMCSF
  • Herpes simplex virus e.g., G207, HSV-1716, T-VEC,
  • Embodiment 392 The kit according to any one of embodiments 384-391, wherein said kit contains both an IDO inhibitor and an ICD inducer.
  • Embodiment 393 The kit of embodiment 392, wherein said IDO inhibitor and said ICD inducer are in separate containers.
  • Embodiment 394 The kit of embodiment 392, wherein said IDO inhibitor and said ICD inducer are in the same container.
  • Embodiment 395 The kit of embodiment 394, wherein said IDO inhibitor and said ICD inducer are provided as a nanoparticle drug carrier according to any one of embodiments 132-195.
  • Embodiment 396 A formulation for inducing immunogenic cell death, said formulation comprising a nanomaterial that induces ICD.
  • Embodiment 397 The formulation of embodiment 396, wherein said formulation comprises a pharmaceutical formulation comprising said nanomaterial that induces ICD and a pharmaceutically acceptable carrier.
  • Embodiment 398 The formulation according to any one of embodiments
  • said formulation is a unit dosage formulation.
  • Embodiment 399 The formulation according to any one of embodiments
  • Embodiment 400 The formulation according to any one of embodiments 396-399, wherein said nanomaterial contains or comprises a nanomaterial that induces ICD.
  • Embodiment 401 The formulation of embodiment 400, wherein said nanomaterial that induces ICD forms a nanoparticle.
  • Embodiment 402 The formulation of embodiment 400, wherein said nanomaterial that induces ICD comprises a core-shell nanoparticle.
  • Embodiment 403 The formulation of embodiment 400, wherein said nanomaterial that induces ICD comprises a doped nanoparticle.
  • Embodiment 404 The formulation according to any one of embodiments
  • said nanomaterial comprises a material selected from the group consisting of CuO, Cu 2 0, Sb 2 0 3 , As 2 C"3, Bi 2 0 3 , P 2 0 3 , ZnO, Ti0 2 , graphene oxide, and 2D materials other than graphene or graphene oxide.
  • Embodiment 405 The formulation of embodiment 404, wherein said nanomaterial comprises CuO.
  • Embodiment 406 The formulation according to any one of embodiments 404-405, wherein said nanomaterial comprises Sb 2 0 3 .
  • Embodiment 407 The formulation according to any one of embodiments
  • nanomaterial comprises ZnO.
  • Embodiment 408 The formulation according to any one of embodiments
  • Embodiment 409 The formulation according to any one of embodiments
  • Embodiment 410 The formulation of embodiment 409, wherein said 2D material comprises a material selected from the group consisting of graphene, graphyne, borophene, germanene, silicene, Si 2 BN, stanene, phosphorene, bismuthene, molybdenite, metals, and a 2D supracrystal.
  • Embodiment 41 1 The formulation of embodiment 410, wherein said nanomaterial comprises graphene oxide.
  • the agent(s) that induce ICD exclude cisplatin, and/or in certain embodiments the agent(s) that induce ICD exclude doxorubicin.
  • the terms "subject,” “individual,” and “patient” may be used interchangeably and refer to humans, as well as non-human mammals (e.g., non-human primates, canines, equines, felines, porcines, bovines, ungulates, lagomorphs, and the like).
  • the subject can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, as an outpatient, or other clinical context.
  • the subject may not be under the care or prescription of a physician or other health worker.
  • a subject in need thereof refers to a subject, as described infra, that suffers from, or is at risk for a cancer as described herein.
  • the subject is a subject with a cancer ⁇ e.g., pancreatic ductal adenocarcinoma (PDAC), breast cancer ⁇ e.g., drug-resistant breast cancer), colon cancer, brain cancer, and the like).
  • PDAC pancreatic ductal adenocarcinoma
  • breast cancer ⁇ e.g., drug-resistant breast cancer
  • colon cancer e.g., brain cancer, and the like.
  • the methods described herein are prophylactic and the subject is one in whom a cancer is to be inhibited or prevented.
  • the subject for prophylaxis is one with a family history of cancer and/or a risk factor for a cancer ⁇ e.g., a genetic risk factor, an environmental exposure, and the like).
  • treat when used with reference to treating, e.g., a pathology or disease refers to the mitigation and/or elimination of one or more symptoms of that pathology or disease, and/or a delay in the progression and/or a reduction in the rate of onset or severity of one or more symptoms of that pathology or disease, and/or the prevention of that pathology or disease.
  • treat can refer to prophylactic treatment which includes a delay in the onset or the prevention of the onset of a pathology or disease.
  • “cotreatment” when used in reference to the coadministration of a first compound (or component) ⁇ e.g., an ICD inducer) and a second compound (or component) ⁇ e.g., an IDO inhibitor) indicates that the first compound (or component) and the second compound (or component) are administered so that there is at least some chronological overlap in the biological activity of first compound and the second compound in the organism to which they are administered.
  • Coadministration can include simultaneous administration or sequential administration. In sequential administration there may even be some substantial delay ⁇ e.g., minutes or even hours) between administration of the first compound and the second compound as long as their biological activities overlap.
  • the coadminstration is over a time frame that permits the first compound and second compound to produce an enhanced therapeutic or prophylactic effect on the organism.
  • the enhanced effect is a synergistic effect.
  • ICD immunogenic cell death
  • cytostatic agents such as anthracyclines (Obeid et al. (2007) Nature Med., 13(1): 54-61), oxaliplatin and bortezomib, or radiotherapy and photodynamic therapy (PDT).
  • immunogenic apoptosis of cancer cells can induce an effective antitumor immune response through activation of dendritic cells (DCs) and consequent activation of specific T cell response (Spisek and Dhodapkar (2007) Cell Cycle, 6(16): 1962-1965).
  • DCs dendritic cells
  • ROS reactive oxygen species
  • ICD In addition to facilitating tumor cell death that facilitates antigen presentation by dendritic cells, ICD is characterized by secretion or release of damage- associated molecular patterns (DAMPs), which exert additional immune adjuvant effects.
  • DAMPs damage- associated molecular patterns
  • Calreticulin (CRT) one of the DAMP molecules, which is normally in the lumen of the ER, is translocated to the surface of dying cell where it functions as an "eat me" signal for phagocytes.
  • Other important surface exposed DAMPs are heat-shock proteins (HSPs), namely HSP70 and HSP90, which under stress condition are also translocated to the plasma membrane.
  • HSPs heat-shock proteins
  • HMGB 1 is considered to be a late apoptotic marker and its release to the extracellular space appears to be required for the optimal release and presentation of tumor antigens to dendritic cells. It binds to several pattern recognition receptors (PRRs) such as Toll-like receptor (TLR) 2 and 4, which are expressed on APCs.
  • PRRs pattern recognition receptors
  • TLR Toll-like receptor
  • ATP binds to purinergic receptors on APCs.
  • IDO inhibitor IDO pathway inhibitor
  • IDO pathway inhibitor IDO pathway inhibitor
  • IDO pathway inhibitor IDO pathway inhibitor
  • IDO pathway inhibitor IDO pathway inhibitor of the IDO pathway
  • agent a molecule or a composition
  • IDO indoleamine-2,3-dioxygenase
  • IDO is an intracellular heme-containing enzyme that initiates the first and rate-limiting step of tryptophan degradation along the kynurenine pathway.
  • the indoleamine 2, 3 -di oxygenase (IDO) pathway regulates immune response by suppressing cytotoxic T cell function, enhancing regulatory T cell activity (Tregs) and enabling tumor immune escape, either at the tumor or regional lympnode sites.
  • An IDO pathway inhibitor can inhibit the IDO enzyme directly or by interfering or perturbing IDO effector pathway components.
  • Such components include, but are not limited to: ID02, tryptophan 2,3-dioxygenase (TDO), the mammalian target of rapamycin (mTOR) pathway, arylhydrocarbon receptor (AhR) pathway, the general control nonderepressible 2 (GCN2) pathway, and the AhR/IL-6 autocrine loop.
  • the nanocarrier comprises a lipid bilayer encasing (or surrounding or enveloping) the porous particle core.
  • the nanoparticle is a porous silica nanoparticle (e.g., mesoporous silica nanoparticle or "MSNP").
  • lipid refers to conventional lipids, phospholipids, cholesterol, chemically functionalized lipids for attachment of PEG and ligands, etc.
  • lipid bilayer or “LB” refers to any double layer of oriented amphipathic lipid molecules in which the hydrocarbon tails face inward to form a continuous non-polar phase.
  • liposome or "lipid vesicle” or “vesicle” are used interchangeably to refer to an aqueous compartment enclosed by a lipid bilayer, as being conventionally defined (see, e.g., Stryer (1981) Biochemistry, 2d Edition, W. H. Freeman & Co., p. 213).
  • a “nanovesicle” refers to a "lipid vesicle” having a diameter (or population of vesicles having a mean diameter) ranging from about 20 nm, or from about 30 nm, or from about 40 nm, or from about 50 nm up to about 500 nm, or up to about 400 nm, or up to about 300 nm, or up to about 200 nm, or up to about 150 nm, or up to about 100 nm, or up to about 80 nm.
  • a nanovesicle has a diameter ranging from about 40 nm up to about 80 nm, or from about 50 nm up to about 70 nm.
  • the lipid bilayer in a lipid vesicle or liposome can be referred to as an "unsupported lipid bilayer” and the lipid vesicle itself (when unloaded) can be referred to as an "empty vesicle”.
  • the lipid bilayer coated on mesoporous silica nanopaticles can be referred to as a "supported lipid bilayer” because the lipid bilayer is located on the surface and supported by a porous particle core.
  • the lipid bilayer can have a thickness ranging from about 6 nm to about 7 nm which includes a 3-4 nm thickness of the hydrophobic core, plus the hydrated hydrophilic head group layers (each about 0.9 nm) plus two partially hydrated regions of about 0.3 nm each.
  • the lipid bilayer surrounding the silica nanoparticle comprises a continuous bilayer or substantially continuous bilayer that effectively encapsulates and seals the nanoparticle.
  • the term "selective targeting” or “specific binding” refers to use of targeting ligands on the surface of a drug delivery nanocamer (e.g., a LB-coated nanoparticle).
  • the targeting ligand(s) are on the the surface of a lipid bilayer of LB-coated nanoparticle.
  • the ligands interact specifically/selectively with receptors or other biomolecular components expressed on the target, e.g., a cell surface of interest.
  • the targeting ligands can include such molecules and/or materials as peptides, antibodies, aptamers, targeting peptides, polysaccharides, and the like.
  • a coated mesoporous silica nanopaticle, having targeting ligands can be referred to as a "targeted nanoparticle or a targeted drug delivery nanocarrier (e.g., LB-coated nanoparticle).
  • the term "about” or “approximately” as used herein refers to being within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. the limitations of the measurement system, i.e. the degree of precision required for a particular purpose, such as a pharmaceutical formulation.
  • “about” can mean within 1 or more than 1 standard deviation, per the practice in the art.
  • “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5% and more preferably still up to 1% of a given value.
  • the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • the term "about” meaning within an acceptable error range for the particular value should be assumed.
  • drug refers to a chemical entity of varying molecular size, small and large, naturally occurring or synthetic, that exhibits a therapeutic effect in animals and humans.
  • a drug may include, but is not limited to, an organic molecule (e.g., a small organic molecule), a therapeutic protein, peptide, antigen, or other biomolecule, an oligonucleotide, an siRNA, a construct encoding CRISPR cas9 components and, optionally one or more guide RNAs, and the like.
  • a "pharmaceutically acceptable carrier” as used herein is defined as any of the standard pharmaceutically acceptable carriers.
  • the pharmaceutical compositions of the subject invention can be formulated according to known methods for preparing
  • the pharmaceutically acceptable carrier can include diluents, adjuvants, and vehicles, as well as carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating material that does not react with the active ingredients of the invention. Examples include, but are not limited to: phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions.
  • the carrier can be a solvent or dispersing medium containing, for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • Formulations are described in a number of sources that are well known and readily available to those skilled in the art.
  • Remington's Pharmaceutical Sciences (Martin E W [1995] Easton Pa., Mack Publishing Company, 19th ed.) describes formulations which can be used in connection with the drug delivery nanocarrier(s) (e.g., LB-coated nanoparticle(s)) described herein.
  • an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of
  • immunoglobulin genes or derived therefrom that is capable of binding (e.g., specifically binding) to a target (e.g., to a target polypeptide).
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • a typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
  • Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2, a dimer of Fab which itself is a light chainjoined to V H -C H 1 by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab') 2 dimer into a Fab' monomer.
  • the Fab' monomer is essentially a Fab with part of the hinge region ⁇ see, Fundamental Immunology, W E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments).
  • antibody as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
  • Certain preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), more preferably single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.
  • the single chain Fv antibody is a covalently linked V H -V L heterodimer which may be expressed from a nucleic acid including V H - and V L - encoding sequences either joined directly or joined by a peptide-encoding linker.
  • the first functional antibody molecules to be expressed on the surface of filamentous phage were single-chain Fv's (scFv), however, alternative expression strategies have also been successful.
  • scFv single-chain Fv's
  • Fab molecules can be displayed on a phage if one of the chains (heavy or light) is fused to g3 capsid protein and the complementary chain exported to the periplasm as a soluble molecule.
  • the two chains can be encoded on the same or on different replicons; the important point is that the two antibody chains in each Fab molecule assemble post- translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to, e.g., g3p (see, e.g., U.S. Patent No: 5733743).
  • the scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three- dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (see e.g., U.S. Patent Nos.
  • antibodies should include all that have been displayed on phage (e.g., scFv, Fv, Fab and disulfide linked Fv (see, e.g, Reiter et al. (1995) Protein Eng. 8: 1323-1331) as well as affibodies, unibodies, and the like.
  • phage e.g., scFv, Fv, Fab and disulfide linked Fv (see, e.g, Reiter et al. (1995) Protein Eng. 8: 1323-1331) as well as affibodies, unibodies, and the like.
  • Two-dimensional materials are materials that do not require a substrate to exist. In other words, they can be isolated as freestanding one atom thick sheets. As a practical matter, this definition can be relaxed to include materials with a thickness of a few atoms (e.g., less than about 10 atoms).
  • substantially pure isomer refers to a formulation or composition wherein among various isomers of a compound a single isomer is present at 70%, or greater or at 80% or greater, or at 90% or greater, or at 95% or greater, or at 98% or greater, or at 99%) or greater, or said compound or composition comprises only a single isomer of the compound.
  • a “bioreactive nanomaterial” refers to an engineered biomaterial that induces or catalyzes a biological response.
  • the nanomaterial induces a response by virtue of one or more properties selected from the group consisting of composition, size, shape, aspect ratio, dissolution, electronic, redox, surface display, surface coating, hydrophobic, hydrophilic, an atomically thin nanosheet, or functionalized surface groups" to catalyze the biological response at various nano/bio interfaces.
  • the bioreactive nanomaterial has the ability to induce ICD biological responses in cells (e.g., in tumor cells) and/or as well as activating the innate immune system through delivery of "danger signal” and adjuvant effects.
  • FIG. 1 provides a schematic explaining immunogenic cell death and synergy with an IDO inhibitor (indoximod).
  • DOX delivery to the primary BC site induce a form of stress-induced cell death, characterized by as CRT expression (an "eat-me” signal for dendritic cell uptake), as well as the release of the adjuvant stimuli, HMGB-1 and ATP.
  • CRT expression an "eat-me” signal for dendritic cell uptake
  • HMGB-1 and ATP the release of the adjuvant stimuli
  • the ability of maturating dendritic cells to cross-present endogenous tumor antigens to naive CD8+ T-cells induces the development of cytotoxic T-cells.
  • CTLs are capable of inducing perforin and granzyme B induced death of primary and metastatic cancer cells.
  • indoximod enhances the ICD effect (increased autophagy and ATP production), in addition to interfering in the metabolic immunosuppressive IDO pathway. This induces a synergistic innate and cognitive immune response at the cancer site, and also assists long- lived memory T cell development.
  • Figure 2 illustrates the structure of indoximod and various other IDO pathway inhibitors.
  • Figure 3 illustrates representative examples to show the use of an ester bond to make IDO inhibitor (e.g., indoximod) pro-drug conjugates.
  • IDO inhibitor e.g., indoximod
  • the H 2 group highlighted by red circle
  • the -COOH (green box) in indoximod can then robustly react with the -OH (blue box) in PHGP, Vitamin E or cholesterol, leading to a list of pro-drugs, that can self-assemble as vesicles (or micellar structures) in aqueous solution. It can also be used in the lipid mixture for MS P coating.
  • Figure 4 illustrates representative examples to show the combined use of HO- linker and ester bond to make IDO inhibitor (e.g., indoximod) pro-drug conjugates.
  • IDO inhibitor e.g., indoximod
  • the NH 2 group (highlighted by red circle) is protected in the indoximod before the conjugation reaction.
  • the -COOH (green box) in indoximod can robustly react with one -OH group (blue box) in the linker compound, which can also readily react with -COOH in the oleic acid or DHA molecule via the other -OH group.
  • Figure 5 A shows construction of an IND nanovesicle by self-assembly of
  • Figure 5B whows CryoEM images of DOX/IND-NVs versus commercial DOXIL® liposome.
  • Figure 6 illustrates the synthesis of DOX-laden IND-PL coated MSNP.
  • Figure 7 illustrates western blot assays showing the enhanced effect of IND-
  • Figures 8A-8D illustrate a biodistribution study in a 4T1 orthotopic model.
  • Fig. 8A IVIS imaging to look at primary tumor burden by bioluminescence.
  • Fig. 8B shows the same model shown in Fig. 8 A after receiving IV injection of free Dox, DOXIL® and Dox/IND-NV at identical Dox dose (5mg/kg).
  • Fig. 8C shows results of a separate PK study in which a single IV injection of free Dox, DOXIL® or Dox/IND-NV (Dox 5 mg/kg) was carried out.
  • Fig. 8D shows indoximod concentration measured using UPLC.
  • Figures 9 A and 9B shows ongoing anti-cancer efficacy data in 4T1 orthotopic breast cancer bearing mice.
  • Fig. 9B In fact, in the experiment shown in (Fig. 9A), we also included additional treatment using free Dox plus anti-PDl with a view to demonstrate the advantage of Dox/IND-NV versus a standard chemo/immuno combination therapy in breast cancer.
  • FIG. 10 Panel 10, panels A-C, illustrates the use of a vaccination approach to identify ICD inducers. Consensus guidelines were used to identify DOX and PTX as ICD introducing chemo in vitro and in vivo. Panel A: CRT surface expression was detected flow cytometry, using the indicated drugs at different doses over 24 hr. Screening for FDVIGBl and ATP yielded similar results. Panel B: Animal vaccination study using 2 rounds of subcutaneous injection of dying 4T1 cells one week apart, followed by injecting live cells SC on the contralateral side. Panel C: Spaghetti curves showing interference in 4T1 tumor growth by vaccinating with cells undergoing ICD by DOX and PTX.
  • FIG. 11 panels A-D, illustrates the synthesis, characterization and PK assessment of a DOX/IOND liposome (vesicle).
  • Panel A depicts synthesis of the DOX and IND-PL liposome. Schematic preparation procedures that pinpoints each steps utilized for coining the DOX/IND-Liposome via remote loading approach. Briefly, a lipid film comprising IND-PL, Cholesterol, and DSPE-PEG2K was obtainned by removing the organic solvent using rotary evaporator, which was hydrated in protonating agent, (NH4)2S04 followed by active DOX remote loading. The box in panel A summarizes the IND-PL self- assembly and active DOX loading.
  • Panel B Cartoon showing the self-assembly of DOX-laden IND-Liposome; high magnification cryoEM shows the clear bilayer structure with a chunk of (DOX- H 3 ) 2 S0 4 precipitate presented in the empty core.
  • Neutral DOX is a weak basic molecule that can diffuse across the IND-PL lipid bilayer to the empty inner core of IND-Liposome, where it met with the released free protons NH 4 +, and S0 4 2" from prepackaged (NH 4 ) 2 S0 4 , which converted DOX to (DOX-NH 3 ) 2 S0 4 precipitate. Upon formation of precipitate, DOX was unable to cross back the lipid bilayer.
  • Panel C Side by side comparison of DOX/IND-Liposome and DOXIL® in terms of DOX and IND loading, size, polydispersity, charge and the endotoxin level.
  • Panel D DOX precipitates as crystals in the nanovesicle, with identical morphology to DOXIL® as shown in the CryoEM picture. DLS sizes of DOX/IND-liposome is very similar to DOXIL®.
  • Figure 12 panels A-E, illustrates the biodistribution and PK assessment of a
  • DOX/IND liposome achieved comparable pharmacokinetics (PK) and tumor uptake as DOXIL®, which are much improved compared to free DOX.
  • Panel A Establishment of syngeneic 4T1 orthotopic model that will be used for PK, biodistribution and efficacy study. Autopsy and IVIS images of the 4T1 -derived orthotopic BC model in immunocompetent Balb/c mice. Briefly, luciferase-transfected 4T1 BC cells (1 million in 1/1, v/v, matrigel/DMEM) were injected to the 2nd mammary fat pad of Balb/c mice.
  • DOX MTD DOX MTD and rationale for choosing the proper and clinically relevant DOX dose.
  • the MTD for DOX, DOXIL®, and DOX/IND-Liposome were determined at 8, 15, and 15 mg/kg for respectively.
  • DOXIL® is IV administered at a dose of -50 mg/cm 2 once a month. This allows us to use this formula to convert the human dose into mouse dose.
  • a human DOX dose of 50 mg/cm 2 /month equals to DOX mouse dose of -16.4 mg/kg per month.
  • Figure 13 illustrates the treatments used in the 4T1 orthotopic breast cancer
  • DOXIL® in restraining the orthotopic BC in immunocompetent mice.
  • Panel A DOX/IND- Liposome (#7, DOX: 5 mg/kg, IND dose was at 8.7 mg/kg) was IV injected to mice on days 8, 11, and 14, respectively.
  • Tumor growth inhibition curve showed markedly improved antitumor efficacy using DOX/IND liposome versus controls (**P ⁇ 0.01, ANOVA).
  • IHC showing IDO expression (brown color) at the orthotopic tumor site.
  • Panels B and C Representative tumor images and averaged tumor weights from different groups after sacrificing mice on day 22.
  • Panel D Representative lung metastasis from each group. The quantified bioluminescence intensity displays the drastically reduced tumor lung metastasis in #1 as compared to #3 and other controls (**P ⁇ 0.01, ANOVA), notwithstanding #3 minimized the lung metastasis when compared to other controls (*P ⁇ 0.05, ANOVA).
  • Panel E Dual delivery #7, dramatically prolonged the mice survival as manifested by the Kaplan-Meier analysis (**P ⁇ 0.01, Log- rank Mantel-Cox test).
  • FIG. 15 Panels A-F, shows that combined use of anti-PD-1 immune checkpoint inhibitor with DOX/IND-Liposome led to the further boosted tumor control with completely depleted metastasis.
  • Panel A IHC staining showing the high PD-1 expression in the 4T1 BC tissue, which provides a legitimate reason for combining anti PD-1 therapy.
  • Panel B Tumor volume curve over the time post different treatments, in which combination of anti PD-1 (IP injected at 100 ⁇ g/mouse on day 8, 11, and 14) and DOX/IND-Liposome (# 8) furthered the tumor suppression significantly.
  • Panels C and D Selective dissected tumors from the mice in #1, #6, #7, and #8, and their calculated averaged tumor weights.
  • Panel E Representative lung metastasis images from #1, #6, #7, and #8. Combination of anti PD-1 and DOX/IND-Liposome resulted in complete lung metastasis remission.
  • Panel F The mice survival was further lengthened in #8. Results are expressed as mean ⁇ SEM. *p ⁇ 0.05; **p ⁇ 0.01, (ANOVA).
  • FIG. 16 Panel 16, panels A-D, shows that systemic knockdown of CD8 T cells drastically reduced the antitumor efficacy of DOX/IND-Liposome in 4T1 BC mice model.
  • CD8 + T cells were systemically depleted by IP injecting anti CD8 3 days before the first drug treatments and were continued every 2-3 days until the termination of the study.
  • Panel A Upon injecting the anti-CD8 antibody, the tumor growth delay from DOX/IND- Liposome were prominently decreased.
  • Panel B IVIS imaging showing the representative bioluminescence of the orthotopic 4T1 tumor development, which aligns with the tumor growth curve exhibited in A.
  • Panel C Representative lung metastasis images from #1, #1 and #9, where systemic knockdown of CD8 led to the recovery of lung metastasis.
  • Panel D CD8 depletion also contributed to the drastically lessened mice survival rate. Results are expressed as mean ⁇ SEM. **p ⁇ 0.01, (ANOVA).
  • FIG. 17 Panel 17, panels A-F, shows that the DOX/IND-liposome antitumor effect involves the activation of both innate and adaptive immunity in breast cancer.
  • a panel of immune biomarkers was comprehensively evaluated in the tumors from the orthotopic 4T1 tumor model shown in Table 13 (Example 3).
  • Panel B Multi-color flow cytometry analysis for innate immune markers: CD91 + DC-like cells (CD45 + CD1 lb + CDl lc + CD91 + ),
  • CD80 + /CD86 + DCs CD45 + CD1 lc + CD80 + CD86 + ), and CD103 + DCs
  • Dual delivery #7 significantly improved the CD8/Tregs ratios, IFN-y + and Granzyme B + T cells, and CD91 + , CD80 + /CD86 + , and CD103 + DCs, particularly when combined with anti PD-1 immune checkpoint blockade.
  • Panel F Western blotting of P-S6K in tumors in the groups treated with IND-Liposome. Elevated P-S6K intensity indicates the enhanced mTOR stimulation (left).
  • Figure 18 shows the results of a safety assessment of DOX/IND liposome in mice. Assessment of blood chemistry to reveal the safety of different DOX formulations. Free DOX-bearing groups had severe toxicity as evidenced by the greatly increased levels of cardiac troponin I, creatine kinase, ALT, AST, and creatinine, while this phenomenon was not seen in dual delivery #7, substantiating its superior safety feature in vivo. Results are expressed as mean ⁇ SEM. *p ⁇ 0.05; **p ⁇ 0.01; #p ⁇ 0.001, (ANOVA). [0487] Figure 19, panels A-D, illustrates in vitro characterization of DOX ICD profile in 4T1 breast cancer cell line.
  • Panel A Flow cytometry analysis to show the normalized CRT expression levels after translocation from endoplasmic to cell surface in 4T1 cells treated with PBS (Ctr), Cis, DOX, PTX and OX at various concentrations for 24 h.
  • Panel B Confocal microscopy depicting the surface induction of CRT, in the presence of Cis (100 ⁇ ), DOX (1 ⁇ ), PTX (1 ⁇ ) and OX (50 ⁇ ) for 24 h, respectively in 4T1 cells. The CRT, cell nuclei, and surface membrane and were detected by ALEXAFLUOR®647- conjugated anti-CRT, Hoechst 33342, Alexa Fluor® 488-Conjugated Wheat Germ
  • Figure 21 panels A-C, illustrates results of a vaccination study using dying
  • Panel C Measurement of CD103 (CD45 + CD1 lb + CDl lc + CD103 + ) and
  • FIG. 22 Panel A-H, illustrates in vivo 4T1 othotopic breast tumor efficacy evaluation as shown in Figure 16.
  • Panel A "Spaghetti plots" detailing each individual mouse tumor growth from different treatment groups.
  • Panel B Representative tumor
  • Panel C Mice weight change monitoring during the animal experiments. Mice were sacrificed on day 22 and tumors were obtained for flow cytometry and IHC analysis (Figure 16). IHC staining analysis for tumoral Foxp3 (panel D), CD91 (panel E), CC-3 (panel F), IL12p70 (panel G), and LC-3 (panel H). Results are expressed as mean ⁇ SEM. *p ⁇ 0.05; **p ⁇ 0.01,
  • Panel A Western blotting to determine the phosphorylated S6 kinase (P-S6K) levels in 4T1 cells after being dosed with IND or IND-Liposome at 10 ⁇ and 50 ⁇ , respectively for 3h in tryptophan-deficient DMEM.
  • the IND-Liposome showed significantly enhanced P-S6K induction in a dose dependent fashion as compared to free IND treatment, which is indicative of its superior mTOR pathway stimulation.
  • a quantitative data of the P-S6K bands using image J software was displayed in the right panel based on three independent repeats.
  • Panel B The supernatants from the cell culture in panel A were collected for the analysis of IL-6 release from 4T1 cells.
  • Panel C Intracellular internalization of IND-Liposome in 4T1 cells. Cells were challenged with free IND or IND-Liposome at equivalent 100 ⁇ g/mL IND dose for 4, 24, and 72 h, respectively. Cell suspensions were then collected for UPLC-MS/MS measurement for IND or IND-PL following our established protocol. The data delineate that IND-PL drastically improved intracellular uptake of IND with efficient and rapid release of IND. Results are expressed as mean ⁇ SEM. *p ⁇ 0.05; **p ⁇ 0.01; # p O.001, (ANOVA).
  • Figure 24 shows a schematic to illustrate how contemporaneous delivery of OX and IND, including through the creative use of nanocarriers, could induce effective anti- PDAC immunity.
  • ICD induction of ICD by OX and interference in the IDO pathway by IND could synergize in generating an effective anti-tumor immune response, premised on enhanced tumor cell uptake and antigen presentation by participating DCs, coupled with interference in the immune suppressive effects of IDO in the TME.
  • Figure 25 panels a-g, shows that oxaliplatin-induced ICD provides a successful anti-PDAC vaccination approach.
  • Panel a Confocal microscopy showing the induction of the ICD marker, CRT, in the presence of PBS, Cis (100 ⁇ ), OX (50 ⁇ ), and DOX (1 ⁇ ) for 4 h in KPC cells.
  • the cell nuclei, surface membrane and CRT were detected by Hoechst 33342, Alexa Fluor® 488-Conjugated Wheat Germ Agglutinin, and Alexa Fluor®647-conjugated anti-CRT antibody staining, respectively.
  • Panel b CRT surface detection by flow cytometry, using the same conditions and reagents as in (a).
  • Panel c Animal experimentation using 2 rounds of vaccination one week apart, followed by injecting live KPC cells SC on the contralateral side.
  • Tumors were harvested on day 29 for IHC and flow cytometry analysis.
  • Panel d Spaghetti curves to show KPC tumor growth in the contralateral flank.
  • Panel e Tumor harvesting was performed after animal sacrifice to conduct IHC. Representative images are shown for the IHC staining of CD8 (upper panel) and Foxp3 (lower panel) T cells. The tumor tissues were also analyzed by flow cytometry to determine the CD8/Tregs ratio (see experimental section for details) (right panel).
  • Panel f IHC staining for cleaved caspase-3 (CC-3) and IFN- ⁇ to demonstrate recruitment of cytotoxic T cells in response to ICD.
  • Panel g The 3 surviving animals in the OX-treated group, described in panel c, received orthotopic implant of live KPC cells on day 74. Animals maintained their tumor free status up to 132 days, whereupon they were sacrificed for harvesting of immune splenocytes to perform an adoptive transfer experiment. IV injection of the immune splenocytes into the tail vein of B6/129 mice prevented the growth of KPC cells, implanted SC. The controls included IV administration of non-immune splenocytes or saline. The same experiment was also carried out in mice receiving SC injection of B16 melanoma cells.
  • FIG. 26 panels a-f, illustrates synthesis of a self-assembling indoximod
  • Panel a Synthesis route for generating the phospholipid-conjugated IND prodrug (IND-PL). Detailed characterization data are provided in Fig. 34.
  • Panel b Illustration depicting self-assembly of IND-PL nanovesicles (IND-NV), with IND securely anchored in the lipid bilayer. We also show a representative cryoEM image of the spherical IND-NV, with diameter -80 nm and lipid bilayer thickness of ⁇ 7 nm. A lower magnification EM picture is shown in Fig. 34, panel h.
  • Panel c UPLC -MS/MS to determine the cellular uptake and release of IND-PL.
  • KPC cells were treated with 100 ⁇ g/mL free IND or IND-NV for 4, 24, or 72 h, respectively.
  • the data show the fold-increase of the intracellular drug concentration as compared to free IND.
  • a typical UPLC-MS/MS readout is shown in Fig. 35. The detailed sample preparation and analysis are described in Fig. 35.
  • Panel d Role of IDO-controlled signaling pathways in PDAC immune suppression, and the effect of IND inhibition or perturbation of these pathways (red arrows).
  • mTOR mammalian target of rapamycin
  • P-S6K phosphorylated S6 kinase
  • AHR aryl hydrocarbon receptor
  • Kyn Kynurenine
  • GNC2 - 'general control non-derepressible 2 Panel e: KPC cells were treated with free IND or IND-NV at the indicated concentrations for 3 h. Western blot assays showing the enhanced effect of IND-PL on mTOR signaling, leading to the phosphorylation and activation of P-S6 (upper panel). Western blotting also showed enhanced inhibition of AHR expression by the conjugated vs. free drug (lower panel).
  • Panel f Assessment of IL-6 release into the supernatant by ELISA, demonstrating the enhanced suppressive effect of IND-NV over free drug. Results are expressed as mean ⁇ SEM. *p ⁇ 0.05; **p ⁇ 0.01.
  • FIG. 27 panels a-g, shows that co-administration of IND-NV with OX at the tumor site augments anti-PDAC immunity.
  • Panel a IT co-administration into tumors established by SC injection of KPC cells in syngeneic mice. Treatment details are provided in the methods section.
  • Panel b KPC tumor growth curve after a single IT injection of the various drugs, when the tumor size approached 60-80 mm 3 . OX was injected at 1.25 mg/kg. Low (L, 2.5 mg IND /kg) and High (H, 12.5 mg IND /kg) refer to the IND or IND-NV doses.
  • Panel c Representative tumor images from each group after animal sacrifice on day 31.
  • Panel d IHC depicting CD8 and Foxp3 biomarkers in harvested tumor tissue. The full panels of IHC staining data are shown in Fig. 36, panels a-j .
  • Panel e Flow cytometry determination of CD8/Tregs ratio, as described in Fig. 26, panel e.
  • Panel f Flow cytometry analysis to determine CD91 expression in the population of CD45 + /CD1 lb + /CDl lc + cells in the tumor tissue.
  • Panel g IHC to depict CRT and HMGB-1 expression in the harvested tumor tissues. Results are expressed as mean ⁇ SEM. *p ⁇ 0.05; **p ⁇ 0.01.
  • FIG. 28 panels a-d, illustrate the development of a dual delivery carrier for OX plus IND using lipid-bilayer coated mesoporous silica nanoparticles (OX/IND-MSNP).
  • Panel a Schematic to show the structure of OX-laden MSNP, in which the drug is trapped by a lipid bilayer containing IND-PL. This leads to stable entrapment of OX in the pores, with IND-PL trapped in the bilayer. The coating procedure provides uniform and instantaneous sealing of all particle pores.
  • the development of an optimized lipid coating mixture (75% IND-PL, 20% cholesterol and 5% DSPE-PEG2K), is described in Fig. 37, panel a.
  • the CryoEM picture shows a spherical MSNP core and the coated lipid bilayer.
  • Low- magnification cryoEM images are provided in Fig. 37, panel b.
  • Dylight 680-labeled DMPE was used in the lipid bilayer for NIR imaging.
  • Ex vivo imaging was performed for tumor, heart, liver, spleen, kidney, and lung tissue harvested from the animals 24 and 48 h post injection.
  • FIG. 29 shows that dual delivery of OX plus IND-NV by MSNP induced effective anti-PDAC immunity in the orthotopic tumor model.
  • Panel b Representative IVIS imaging on days 10, 18, 27, and 36, according to which the normalized tumor burden was plotted as fold-increase compared to the non-treated control.
  • Panel c Representative ex vivo bioluminescence imaging on day 36 to show the effect of treatment on metastatic tumor spread to the stomach, intestines, liver, spleen, kidneys, diaphragm, and abdominal wall, but not the heart or lung.
  • treatment with anti-CD8 and anti-TLR4 antibodies, as well as an injectable siRNA for knockdown of CD91 The effect of interference in the immune response is shown in Fig. 41, panel a.
  • Panel e Serum amylase levels as a reflection of the effect of effective tumor shrinkage by OX/IND-MSNP.
  • Panel f IHC staining for CD8 + and Foxp3 + T cells in tumor tissue, collected in (c), (left panel).
  • FIG. 30 Panel 30, panels a-b, illustrates immuno-PET imaging to demonstrate the induction of the systemic immune response by OX/IND-MSNP administration to animals carrying orthotopic KPC tumors.
  • Panel b To evaluate the CD8 + signal at the tumor site, the operator-defined ROIs were used to demonstrate a 6.2- and 7.5-fold increase in the signal intensity in the tumor interior and periphery, respectively, during OX/IND-MSNP compared to saline treatment. Results are expressed as mean ⁇ SEM. *p ⁇ 0.05; **p ⁇ 0.01.
  • Figure 31 shows flow cytometry analysis to show the normalized
  • Figure 31 panel b shows the same flow cytometry analysis in PANC-1 cells treated for 4 or 24 h.
  • Figure 31, panel c shows ELISA measurement of HMGB-1 release from KPC and PANC-1 cells after treatment with Cis, OX, and DOX for 4 h. *p ⁇ 0.05; **p ⁇ 0.01.
  • Figure 32, panel a shows (IVIS optical imaging to follow the tumor burden in the vaccination experiment, as described in Fig. 25, panel d.
  • Figure 32, panel b shows monitoring of animal weight in the vaccination experiment.
  • Figure 32, panel c shows IHC analysis to discern CD4 expression in harvested tumor tissue on day 29.
  • FIG. 34 panels a-i, shows detailed characterization of the intermediary products during IND-PL synthesis, as shown in Fig. 26, panel a.
  • the table in panel a summarizes the intermediates and show the yield at each stage of the synthesis process.
  • Panels, b, and c show ESI-MS, 1H-NMR and 13 C-NMR data for N-Boc-IND.
  • Panels d and e show N-Boc-IND-PL analysis by ESI-MS, 1H-NMR and 13 C-NMR.
  • Panels f and g show IND-PL analysis by ESI-MS, 1H-NMR and 13 C-NMR.
  • Panel h shows low magnification cryoEM image of IND-NV.
  • Panel I shows the unfavorable PK of free IND, as demonstrated by the short half-life and low tumor retention capability, and highlights the necessity of a nano-enabled approach to improve retention of the IDO inhibitor.
  • Figure 35, panel a shows UPLC-MS/MS analysis of the cellular content and intracellular release of IND from IND-NV.
  • Figure 35, panel b shows that the establishment of distinct diluting times for IND (1.23 min), PL (3.97 min), and IND-PL (3.08 min) allowed us to use UPLC analysis to demonstrate the total and released drug content in the cells, as described in Fig. 26, panel c.
  • Figure 35, panel c shows the results of an abiotic experiment performed to demonstrate that acidic pH and esterase activity can release IND from the prodrug.
  • FIG. 37 panel a, illustrates the development and optimization of the IND-PL biofilm to coat MS P with a lipid bilayer. This was accomplished by adjusting the ratio of IND-PL vs. cholesterol and DSPE-PEG2K in the lipid mixture. The optimal molar ratio for IND-PL/Cholesterol/DSPE-PEG2K was determined to be 75:20:5 (ratio #4).
  • Panel b shows nanoparticle characterization of OX/IND-MSNP in DI water, PBS, and DMEM containing 10% FBS on days 1 and 30. OX and IND loading capacities were determined to be 4.4% and 44.3%), respectively. The particles had negligible endotoxin content.
  • Figure 38 shows ICP-OES analysis to quantify the Si content in the orthotopic tumor experiment shown in Fig. 28, panel c.
  • Figure 39, panel a shows representative autopsy images of the animals treated with the dual delivery MSNP, as described in Fig. 29, panel c. The broken lines highlight the primary tumors and their local invasion, while the arrows point to metastases.
  • Figure 39, panel b shows a heat map summary of the ex vivo imaging data after tumor and organ harvesting for the experiment shown in Fig. 29, panel c. This demonstrates a significant reduction in tumor metastases in response to OX/IND-MSNP treatment, and to a lesser extent OX-MSNP.
  • Figure 40 shows the full panel of IHC data collected during the systemic delivery experiment in Fig. 29, showing results for the following markers: CD8 (panel a), Foxp3 (panel b), CRT (panel c), CD91 (panel d, left panel), HMGB-1 (e), TLR4 (panel f, left panel), IFN- ⁇ (panel g), perforin (panel h), IL-10 (panel i), and CC-3 (panel j).
  • FIG. 41 Panel a-d, shows a demonstration of the impact of IV injection of antibodies to CD8 and TLR-4, or an injectable pool of siRNAs targeting CD91 in the systemic biodistribution experiment described in Fig. 29.
  • OX/IND-MSNP 5 mg/kg OX and 50 mg/kg IND
  • anti-CD8, anti-TLR4 or CD91 siRNA knockdown 7.
  • the normalized tumor burden as reflected by the luminescence intensity in the ROI, was plotted and displayed in the right side panel.
  • Panel b Representative autopsy results and ex vivo bioluminescence imaging intensity is depicted to show the impact of interference in the immune response on tumor growth and metastases.
  • Panel c Animal survival rate in the same experiment.
  • Panel d Impact on the CD8/Treg ratio in the same experiment.
  • Figure 42 panel A shows monitoring of animal weight during treatment with the dual delivery carrier in Fig. 29.
  • Fig. 42, panel B shows monitoring of liver enzymes (ALT, AST, and ALP) in the orthotopic tumor model IV described in Figs. 29 and 41.
  • OX/IND-MSNP dual delivery particles did not exert toxicity during the experiment. Instead, it helped to protect against liver toxicity from IV injected free OX. Similarly, there was no biochemical evidence of toxicity in the kidney or heart (data not shown). **p ⁇ 0.01.
  • Figure 43 shows normalized area of interest (ROI) scanning data to show the radio label portioning to the spleen and tumor draining lymph nodes (TDLN)in mice treated by saline, OX-MSNP, and OX/IND-MSN,P followed by IV immuno-PET particle (89Zr- malDFO-169 cDb) injection, as described in Fig. 30.
  • Figures 44A-44C illustrate the screening of nanomaterial (NM)-induced immunogenic cell death (ICD) in KPC pancreatic cancer cell after 24 h treatment with engineered nanoparticles.
  • NM nanomaterial
  • ICD immunogenic cell death
  • Figure 44A illustrates the induction of calreticulin (CRT) following treatment with a nanomaterial ⁇ see, e.g., Table 10 in Example 7).
  • Figure 44B shows dose and time-dependent CRT induction in KPC cells.
  • Figure 44C shows the high mobility group box 1 protein (FDVIGB-1) concentration in the supernatant of the KPC cells after being treated with various NMs.
  • Figures 45 shows cytotoxicity profile of metal oxides (MOs), graphene oxides
  • FIGs 46A and 46B show the results of a vaccination experiment using metal and metal oxide.
  • Fig. 46A Animal experimentation using 2 rounds of vaccination (dying KPC cells treated with metal oxide nanoparticles) one week apart, followed by injecting live KPC cells SC on the contralateral side. Spaghetti curves to show KPC tumor growth in the contralateral flank.
  • Fig. 46B IVIS imaging to monitor the tumor growth on the contralateral flank of mice shown in Fig. 46A. At the conclusion stage, the tumor tissues were used for flow cytometry experiment to measure CD8/Treg cell ratios.
  • FIG. 47A-47C show the results of an intratumoral injection (IT) experiment using metal and metal oxide nanoparticles.
  • KPC cells were subcutaneously injected into B6/129 mice.
  • Fig. 47A Dose-seeking experiment for CuO nanoparticle.
  • the subQ tumors received single IT injection of CuO nanoparticle at 15, 30, 50 and 100 ug/mouse. The tumors were monitored up to 23 days.
  • Fig. 47B In a pilot efficacy study using IT injection, KPC subQ tumor mice received single IT injection using indicated NMs. The doses were shown in the figure. Tumor growth was monitored up to -23 days.
  • Fig. 47C On day 23, the tumors were harvested and single cell suspension was collected for flow analysis of various immune biomarkers. Significantly enhanced antitumor immunity was found in CuO group as confirmed by the boosted
  • FIGs 48, panels A-E shows the results of an intratumoral injection (IT) experiment using GOs.
  • Panel A Schematically illustrates the vaccination protocol (similar vaccination study in Fig. 46). KPC cells were treated with GOs. The dying cells were used to vaccinate the mice.
  • Panel B Tumor volume as a function of time pose live KPC for implantation.
  • Panel C IVIS imaging to monitor the KPC tumor growth on the contralateral flank of the mice.
  • Panel D The CD8/Treg cell ratio determined by flow cytometry.
  • Figures 49A-49C show the results of an intratumoral injection (IT) experiment using GOs.
  • Figure 49A KPC subQ tumor mice received single IT injection using indicated GOs. The doses were shown in the figure. Tumor growth was monitored up to -23 days.
  • Figure 49B At the conclusion stage, the tumor samples were harvested for CD8/Treg ratio measurement by flow cytometry.
  • Figure 49C IP of anti-CD8 mAb (200 ug/mouse) interferes the ICD-mediated tumor inhibition induced by small GO.
  • Figure 50 illustrates mass spectrometry of 1-L-MT-PL.
  • Figure 51 illustrates the intracellular uptake of 1 -L-MT-PL in KPC cells at varied time points. UPLC-MS/MS was performed to determine the cellular uptake and release of 1-L-MT-PL. KPC cells were treated with 100 ⁇ g/mL nanovesicles, formed by 1-L- MT-PL. Controls include free 1-L-MT, free 1-D-MT and nanovesicles made by 1-D-MT-PL. After indicated incubation period, the cells were collected via trypsinization and drug extraction, as described by Lu et al. (2017) Nat. Comm. 8: 1811). The data show the fold- increase of the intracellular drug concentration as compared to free 1-L-MT.
  • Figure 52 illustrates western blot and ELISA for P-S6K (cell lysate) and IL-6
  • IL6/STAT3/AHR autocrine signaling loop using 1-L-MT-PL, similar to 1-D-MT-PL.
  • Figure 53 illustrates the effect of different isomers in an in vitro IDO enzymatic assay in 4T1 breast cancer cells.
  • the experiment procedure is similar to literature (Hou et al. (2007) Cancer Res. 67(2): 792-801).
  • lxlO 5 4T1 cells were seeded into 24-well plate. After cell attachment, the cells were treated with different 1-MT isomers or prodrugs at various concentrations. Meanwhile, 100 ng/ml of mouse recombinant IFN- ⁇ was added per well to stimulate IDO expression. Then, the plates were incubated for 20 hr at 37°C in a humidified C0 2 incubator. Supernatants were harvested and analyzed for kynurenine by ELISA. The anti-cancer efficacy experiments were performed in vivo.
  • ICD immunogenic cell death
  • a first treatment modality involves combination of an ICD inducer ⁇ e.g., oxaliplatin) in combination with an IDO inhibitor ⁇ e.g., indoximod) into a single nanocarrier that allows systemic (or local) biodistribution and drug delivery to tumor sites.
  • the dual- delivery approach achieved synergistic enhancement of adaptive and innate immunity ⁇ e.g., anti-PDAC immunity), leading to a significant improvement in animal survival.
  • the nanocarrier comprises a vesicle ⁇ i.e., a lipid bilayer enclosing a fluid).
  • the nanocarrier comprises a nanoparticle ⁇ e.g., a mesoporous silica nanoparticle (MSNP) surrounded (encapusulated) by a lipid bilayer.
  • MSNP mesoporous silica nanoparticle
  • a second treatment modality involves local delivery to a tumor or peri- tumoral region, of an agent that induces ICD ⁇ e.g., oxaliplatin) in combination with a lipid
  • a nanovesicle that comprises an inhibitor of the IDO pathway ⁇ e.g., indoximod.
  • ICD inducer in combination with an IDO inhibitor induces recruitment of cytotoxic CD8 + lymphocytes, depletion of Tregs, reversal of the CD8 + /Foxp3 + ratio, cytotoxic tumor killing, and tumor shrinkage at the local site.
  • IDO inhibitor induces recruitment of cytotoxic CD8 + lymphocytes, depletion of Tregs, reversal of the CD8 + /Foxp3 + ratio, cytotoxic tumor killing, and tumor shrinkage at the local site.
  • These adaptive immune responses were accompanied by boosting of the innate immune system, as reflected by CRT and HMGB1 expression, as well as the activation of a DC population, particularly well-suited for generating cytotoxic T cell responses.
  • a third treatment modality involves vaccination utilizing dying cancer cells
  • Approach 1 Systemic treatment of a cancer by combined delivery of ICD and IDO inhibition.
  • the first approach approach combines an ICD-inducer (e.g., doxirubicin, oxaliplatin, etc.) and an inhibitor of the IDO pathway (e.g., indoximod) into a single nanocarrier, that can provide systemic biodistribution and drug delivery to orthotopic tumor sites.
  • ICD-inducer e.g., doxirubicin, oxaliplatin, etc.
  • IDO pathway e.g., indoximod
  • this dual-delivery approach involves the formation of lipid vesicles where a component of the lipid bilayer comprising the vesicle incorporates or is conjugated to an inhibitor of the IDO pathway (e.g., an indoximod prodrug such as IND-PL) and the vesicle contains an ICD inducer (e.g., doxorubicin (DOX)).
  • an inhibitor of the IDO pathway e.g., an indoximod prodrug such as IND-PL
  • ICD inducer e.g., doxorubicin (DOX)
  • the nanocarrier comprises a mesoporous silica nanoparticle (MS P) containing the ICD inducer (e.g., oxaliplatin) where the silica nanoparticle is surrounded by (encapsulated by ) a lipid bilayer containing (or conjugated to) an IDO inhibitor (e.g., indoximod provided as the prodrug IND- PL (Formula I)).
  • the lipid bilayer (LB) coated MSNP also known as a silicasome (see, e.g., PCT Patent Application No: PCT/US2017/012625) provides effective dual delivery of the ICD inducer and IDO inhibitor.
  • this dual-delivery approach achieved synergistic enhancement of adaptive and innate anti-PDAC immunity, leading to a significant improvement in animal survival.
  • nanomaterials e.g., CuO, Cu 2 0, Sb 2 0 3 , As 2 0 3 , Bi 2 0 3 , P 2 0 3 , ZnO, Ti0 2 , graphene oxide, 2D materials other than graphene or graphene oxide (e.g., graphene, graphyne, borophene, germanene, silicene, Si 2 BN, stanene, phosphorene, bismuthene, molybdenite, metals, 2D supracrystals, and the like), can induce immunogenic cell death (ICD) (see, e.g., Example 7).
  • ICD immunogenic cell death
  • Nanoparticles formed from these ICD inducers can readily be coated with a lipid that contains (or is conjugated to) an IDO inhibitor (e.g., indoximod provided as the prodrug IND- PL (Formula I)).
  • an IDO inhibitor e.g., indoximod provided as the prodrug IND- PL (Formula I)
  • the lipid coated nanomaterial thus forms a dual delivery vehicle for delivery of both an ICD-inducer and an IDO-inhibitor.
  • the following dual-delivery vehicles are contemplated herein: [0531] 1) ICD-inducer/IDO-inhibitor vesicle;
  • any of these carriers are suitable for local treatment of a tumor.
  • any of these carriers can be administered topically (e.g., for skin tumors), or directly, e.g., to an intra-tumoral or peri -tumoral site, e.g., via injection or during a surgical procedure.
  • Dual-Delivery Lipid Vesicles e.g. , ICD IDO inhibitorVesicles
  • dual-delviery nanovesicles are provided for the delivery of an ICD-inducer in combination with an inhibitor of the IDO pathway and/or for the delivery of an ICD inducer and a pharmacological agent other than an ICD inducer or in combination with an ICD inducer in addition to the inhibitor of the IDO pathway.
  • a nanovesicle drug carrier for the combined delivery of an inhibitor of an IDO pathway and an inducer of immunogenic cell death (ICD), where the nanovesicle drug carrier comprises a lipid vesicle where a lipid bilayer effectively forms a vesicle in an aqueous solution, and the lipid or lipid formuation comprising the vesicle is associated with (or conjugated to) an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway (IDO pathway inhibitor); and a cargo within the vesicle where the cargo comprises an agent that induces immunogenic cell death (ICD) (ICD- inducer).
  • ICD immunogenic cell death
  • the lipid vesicle is typically formed from a lipid bilayer.
  • a lipid micelle (which does not comprises a lipid bilayer) is contemplated.
  • a lipid micelle can be comprise a phospholipid prodrug ⁇ e.g., lipid-IDO pathway inhibitor conjugate) and a cargo (typically a lipophilic) cargo can be disposed inside the micelle.
  • the nanovesicle provides an IDO inhibitor and an ICD inducer that are synergistic in their activity against a cancer.
  • the nanovesicle drug carrier when administered systemically, delivers an amount of an ICD inducer effective to induce or to facilitate induction of immunogenic cell death of cancer cells at the tumor site. In certain embodiments the nanovesicle drug carrier, when administered systemically, delivers an amount of IDO inhibitor to partially or fully inhibit an IDO pathway at a cancer site.
  • the inhibitor of the IDO pathway comprises an agent selected from the group consisting of 1-methyl-D-tiyptophan (indoximod, D-1MT), L-1MT, methylthiohydantoin-dl -tryptophan (MTH-Trp, Necrostatin), ⁇ -carbolines (e.g., 3-butyl-P- carboline), naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassinin, N- [2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S- methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl-
  • the IDO inhibitor comprises indoximod.
  • the IDO inhibitor comprises substantially pure "L” indoximod or substantially pure “R” indoximod, or a racemic mixture of "D" and “L” indoximod.
  • the inhibitor of the IDO pathway is disposed in a lipid comprising the vesicle and/or conjugated to a lipid comprising said vesicle.
  • the vesicle comprises a phospholipid.
  • the vesicle comprises a phospholipid, and cholesterol (CHOL).
  • the phospholipid comprises a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C 12-C20 carbon chains.
  • the phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC),
  • the phospholipid comprises a natural lipid selected from the group consisting of egg phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC).
  • the phospholipid comprises an unsaturated fatty acid selected from the group consisting of l,2-dimyristoleoyl-sn-glycero-3- phosphocholine, 1 ,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine, 1 ,2-dioleoyl-sn-glycero-3 - phosphocholine (DOPC), and l,2-dieicosenoyl-sn-glycero-3-phosphocholine.
  • the vesicle comprises an mPEG phospholipid with a phospholipid C14-C18 carbon chain, and a PEG molecular weight ranging from about 350 Da to 5000 Da.
  • the vesicle comprises l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (DSPE-PEG). In certain embodiments the vesicle comprises DPSE-PEG 2 K- In certain embodiments the IDO inhibitor is conjugated to a component of said vesicle. In certain embodiments the IDO inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid. In certain embodiments the IDO inhibitor is conjugated directly to the moiety, while in other embodimetns, the IDO inhibitor is conjugated to the moiety via a linker. In certain embodiments the IDO inhibitor is conjugated to PGHP.
  • the inhibitor of the IDO pathway is conjugated to a phospholipid comprising the lipid vesicle.
  • the inhibitor of the IDO pathway is conjugated to 1- palmitoy l -2-hydroxy-sn-glycero-3-phosphocholine (PL) (e.g., IND-PL, Formula I).
  • PL 1- palmitoy l -2-hydroxy-sn-glycero-3-phosphocholine
  • the bilayered vesicle comprises IND-PL/Chol/DSPE-
  • the vesicle comprises about 75% IND-PL, about 20% cholesterol, and about 5% DSPE-PEG 2 K.
  • the ICD inducer comprises a chemotherapeutic agent selected from the group consisting of doxorubicin, oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
  • the ICD inducer comprises doxorubicin.
  • Dual-Delivery ICD-inducer/IDO-inhibitor
  • LB Coated MSNPs ICD/IDO Silicasomes
  • a dual delivery carrier for an ICD inducer e.g., oxaliplatin
  • an IDO inhibitor e.g., indoximod
  • the carrier comprises lipid-bilayer coated nanoparticles (e.g., mesoporous silica nanoparticles).
  • the IDO inhibitor e.g., indoximod
  • the ICD inducer is provided disposed in and/or conjugated to a component of the lipid bilayer while the ICD inducer is provided on or in (e.g., within the pores) of the nanoparticle, e.g., effectively sealed/contained within the lipid bilayer.
  • the ICD inducer can be provided in or conjugated to the lipid bilayer while the IDO inhibitor is contained on or within the nanoparticle.
  • Such lipid bilayer coated nanoparticle drug delivery systems are capable of delivering two (or more) active agents in precise concentration ratios as desired.
  • the "dual-delivery carrier” comprises indoximod conjugated to a component of the lipid bilayer (e.g., as IND-PL (Formula I)) while the ICD inducer (e.g., oxaliplatin) is disposed within the nanoparticle.
  • the ICD inducer e.g., oxaliplatin
  • the coating, procedure(s) described herein provide uniform and instantaneous sealing of all particle pores (see, e.g., Examples 5 and 2).
  • a nanoparticle drug carrier for the combined delivery of an inhibitor of an IDO pathway and an inducer of immunogenic cell death (ICD)
  • the nanoparticle drug carrier comprises: a mesoporous silica nanoparticle having a surface and defining a plurality of pores that are suitable to receive molecules therein; a lipid bilayer coating the surface; a first cargo comprising an inhibitor of the indoleamine 2,3-dioxygenase (IDO inhibitor); and a second cargo comprising an agent that induces immunogenic cell death (ICD) (ICD-inducer); where the lipid bilayer is substantially continuous and encapsulates the nanoparticle stably sealing the plurality of pores.
  • the nanoparticle drug carrier contains a predefined ratio of IDO inhibitor to ICD-inducer.
  • the IDO inhibitor and the ICD inducer are synergistic in their activity against a cancer (e.g., against PDAC).
  • the drug carrier when administered systemically, is effective to deliver an amount of an ICD inducer effective to initiate or to facilitate induction of immunogenic cell death of a cancer cell.
  • the drug carrier when administered systemically, is effective to deliver an amount of IDO inhibitor to partially or fully inhibit an IDO pathway at a cancer site.
  • the drug carrier can contain/provide a lower dose ICD inducer and/or IDO inhibitor than when these agents are used individually.
  • the combination of the ICD inducer and the IDO inhibitor achieves an anti-cancer activity that cannot be achieved by the use of either agent alone.
  • the IDO inhibitor is disposed in the lipid bilayer and/or conjugated to a lipid comprising said lipid bilayer while the ICD inducer is disposed in the plurality of pores.
  • the ICD-inducer comprises a chemical or biological agent described in Table 2, above.
  • the ICD-inducer comprises a chemotherapeutic agent selected from the group consisting of oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
  • the ICD-inducer comprises oxaliplatin.
  • the ICD inducer comprises an ICD inducing nanomaterial (e.g., CuO, Cu 2 0, Sb 2 0 3 , As 2 0 3 , Bi 2 0 3 , P 2 0 3 , ZnO, Ti0 2 , graphene oxide, 2D materials other than graphene or graphene oxide (e.g., graphene, graphyne, borophene, germanene, silicene, Si 2 BN, stanene, phosphorene, bismuthene, molybdenite, metals, 2D supracrystals, and the like) as described above or in Example 7.
  • the ICD-inducing nanomaterial can be contained on or within the nanoparticle.
  • an ICD-inducing nanomaterial can be coated with a lipid or with a lipid bilayer.
  • the ICD-inducing nanomaterial can incorporate one or more drugs as described herein.
  • the nanomaterial may contain the IDO inhibitor, both of which can be released at a target site (e.g., cancer cell).
  • a target site e.g., cancer cell.
  • nanomaterial comprises graphene oxide, the surface can be functionalized to deliver the IDO- inhibitor.
  • the IDO inhibitor comprises an agent selected from the group consisting of 1-methyl-D-tiyptophan (indoximod, D-1MT), L-1MT,
  • methylthiohydantoin-dl -tryptophan MTH-Trp, Necrostatin
  • ⁇ -carbolines e.g., 3-butyl-P- carboline
  • Naphthoquinone-based e.g., annulin-B
  • S-allyl-brassinin S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S- methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl- brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol
  • the IDO inhibitor comprises an agent shown in Table 3, above. In certain embodiments the IDO inhibitor comprises indoximod.
  • the nanoparticle drug carrier is fabricated so that a population of the drug carriers in suspension shows essentially a substantially unimodal size distribution; and/or shows a PDI less than about 0.2, or less than about 0.1 ; and/or shows a coefficient of variation in size less than about 0.1 or less than about 0.05.
  • the nanoparticle drug carriers distribute to developing tumor sites on IV injection.
  • the nanoparticle drug carrier forms a stable suspension on rehydration after lyophilization.
  • the nanoparticle drug carriers show reduced drug toxicity as compared to free drug and/or drug in liposomes.
  • the nanoparticle drug carrier has colloidal stability in physiological fluids with pH 7.4 and remains monodisperse to allow systemic biodistribution and is capable of entering a disease site by vascular leakage (EPR effect) or transcytosis.
  • nanoparticle e.g., mesoporous silica core
  • lipid bilayer formulations e.g., lipid bilayer formulations, and methods of synthesis are described in the sections below and in the examples.
  • silicasome drug carriers described herein comprise a porous silica (or other material) nanoparticle (e.g., a silica body having a surface and defining a plurality of pores that are suitable to receive molecules therein) coated with a lipid bilayer.
  • the silica nanoparticle can be a mesoporous silica nanoparticle. The fact that the nanoparticle is referred to as a silica nanoparticle does not preclude materials other than silica from also being incorporated within the silica
  • the silica nanoparticle may be substantially spherical with a plurality of pore openings through the surface providing access to the pores.
  • the silica nanoparticle can have shapes other than substantially spherical shapes.
  • the silica nanoparticle can be substantially ovoid, rod-shaped, a substantially regular polygon, an irregular polygon, and the like.
  • the silica nanoparticle comprises a silica body that defines an outer surface between the pore openings, as well as side walls within the pores.
  • the pores can extend through the silica body to another pore opening, or a pore can extend only partially through the silica body such that that it has a bottom surface of defined by the silica body.
  • the silica body is mesoporous. In other embodiments, the silica body is microporous.
  • “mesoporous” means having pores with a diameter between about 2 nm and about 50 nm, while “microporous” means having pores with a diameter smaller than about 2 nm.
  • the pores may be of any size, but in typical embodiments are large enough to contain one or more therapeutic compounds therein. In such embodiments, the pores allow small molecules, for example, therapeutic compounds such as anticancer compounds to adhere or bind to the inside surface of the pores, and to be released from the silica body when used for therapeutic purposes.
  • the pores are substantially cylindrical.
  • the nanoparticles comprise pores having pore diameters between about 1 nm and about 10 nm in diameter or between about 2 nm and about 8 nm. In certain embodiments the nanoparticles comprise pores having pore diameters between about 1 nm and about 6 nm, or between about 2 nm and about 5 nm. Other embodiments include particles having pore diameters less than 2.5 nm. In other words,
  • the pore diameters are between 1.5 and 2.5 nm.
  • Silica nanoparticles having other pore sizes may be prepared, for example, by using different surfactants or swelling agents during the preparation of the silica nanoparticles.
  • the nanoparticles can include particles as large (e.g., average or median diameter (or other characteristic dimension) as about 1000 nm.
  • the nanoparticles are typically less than 500 nm or less than about 300 nm as, in general, particles larger than 300 nm may be less effective in entering living cells or blood vessel fenestrations.
  • the nanoparticles range in size from about 40 nm, or from about 50 nm, or from about 60 nm up to about 100 nm, or up to about 90 nm, or up to about 80 nm, or up to about 70 nm. In certain embodiments the nanoparticles range in size from about 60 nm to about 70 nm. Some embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 1000 nm. Other embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 500 nm. Other embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 200 nm. In some embodiments, are examples of the nanoparticles having an average maximum dimension between about 50 nm and about 1000 nm. Other embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 500 nm. Other embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 200 nm. In some
  • the average maximum dimension is greater than about 20nm, greater than about 30nm, greater than 40nm, or greater than about 50nm.
  • Other embodiments include nanoparticles having an average maximum dimension less than about 500 nm, less than about 300nm, less than about 200nm, less than about 100 nm or less than about 75 nm.
  • the size of the nanoparticle refers to the average or median size of the primary particles, as measured by transmission electron microscopy (TEM) or similar visualization technique.
  • Illustrative mesoporous silica nanoparticles include, but are not limited to
  • MCM-41, MCM-48, and SB A- 15 see, e.g., Katiyaret a/. (2006) J. Chromatog. 1122(1-2): 13-20).
  • mesoporous silica nanoparticles are synthesized by reacting tetraethyl orthosilicate (TEOS) with a template made of micellar rods. The result is a collection of nano-sized spheres or rods that are filled with a regular arrangement of pores. The template can then be removed by washing with a solvent adjusted to the proper pH (see, e.g., Trewyn et al. (2007) Chem. Eng. J. 137(1): 23-29.
  • TEOS tetraethyl orthosilicate
  • mesoporous particles can also be synthesized using a simple sol-gel method (see, e.g., Nandiyanto, et al. (2009) Microporous and Mesoporous Mat. 120(3): 447-453, and the like).
  • tetraethyl orthosilicate can also be used with an additional polymer monomer (as a template).
  • 3-mercaptopropyl)trimethoxysilane (MPTMS) is used instead of TEOS.
  • the mesoporous silica nanoparticles are cores are synthesized by a modification of the sol/gel procedure described by Meng et al.
  • the solution is stirred at 350 rpm at 85°C for 20 min, leading to the formation particles with a primary size of ⁇ 65 nm.
  • the surfactant can be removed by washing the particles with a mixture of methanol/HCl (500: 19 v/v) at room temperature for 24 h.
  • the particles can be centrifuged at 10 000 rpm for 60 min and washed three times in methanol.
  • porous silica nanoparticles e.g., mesoporous silica
  • similar methods can be used with other porous nanoparticles.
  • mesoporous materials that can be used in drug delivery nanoparticles are known to those of skill in the art.
  • mesoporous carbon nanoparticles could be utilized.
  • Mesoporous carbon nanoparticles are well known to those of skill in the art (see, e.g., Huang et al. (2016) Carbon, 101 : 135-142; Zhu et al. (2014) Asian J. Pharm. Sci., 9(2): 82-91; and the like).
  • mesoporous polymeric particles can be utilized.
  • the syntheses of highly ordered mesoporous polymers and carbon frameworks from organic-organic assembly of triblock copolymers with soluble, low-molecular-weight phenolic resin precursors (resols) by an evaporation induced self-assembly strategy have been reported by Meng et al. (2006) Chem. Mat. 6(18): 4447-4464 and in the references cited therein.
  • nanoparticles described herein are illustrative and non-limiting. Using the teachings provided herein numerous other lipid bilayer coated nanoparticles will be available to one of skill in the art.
  • the drug carrier nanoparticles described herein comprise a porous
  • the bilayer composition is optimized to provide a rapid and uniform particle coating, to provide colloidal and circulatory stability, and to provide effective cargo retention, while also permitting a desirable cargo release profile.
  • the lipid bilayer comprises a combination of a phospholipid, cholesterol, and in certain embodiments, a IDO-lipid conjugate, a pegylated lipid (e.g., DSPE-PEG2000), or a factionalized pegylated lipid (e.g., DSPE-PEG2000- maleimide) to facilitate conjugation with targeting or other moieties.
  • a pegylated lipid e.g., DSPE-PEG2000
  • a factionalized pegylated lipid e.g., DSPE-PEG2000- maleimide
  • a coated lipid film procedure can be utilized in which MSNP suspensions are added to a large lipid film surface, coated on, e.g., a round- bottom flask.
  • MSNP suspensions are added to a large lipid film surface, coated on, e.g., a round- bottom flask.
  • lipid bilayer compositions Using different lipid bilayer compositions, a series of experiments can be performed to find a composition and optimal lipid/particle ratio that provides rapid and uniform particle wrapping, coating and effective cargo retention and/or release upon sonication. It is believed that this lipid composition and wrapping cannot be achieved by liposomal fusion to the particle surface under low energy vortexing conditions.
  • the mesoporous silica nanoparticles are coated with a lipid bilayer that incorporates the IDO inhibitor coupled to a lipid (e.g., a phospholipid) or to cholesterol.
  • a lipid e.g., a phospholipid
  • the mesoporous silica nanoparitcles are coated with a lipid bilayer comprising IND-PL, as well as serving to encapsulate oxaliplatin in the porous interior ( Figure 28, panel a).
  • the lipid bilayer formulation described above and in Example 5 is illustrative and non-limiting. Depending on the drug(s) being loaded into the drug delivery carrierand the desired release profile, in various embodiments different lipid bilayer formulations can be used and an optimal formulation can be determined. Thus, while the described molar ratio of 75:20:5 is optimized for the particular combination of oxaliplatin and indoximod, using the methods described in Example 5, the lipid bilayer can routinely be optimized for other combinations of ICD inducer and IDO inhibitor, and lipid bilayer components.
  • the lipid bilayer can comprise: 1) one or more saturated fatty acids with C14-C20 carbon chain, such as
  • DMPC dimyristoylphosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • DAPC diactylphosphatidylcholine
  • One or more unsaturated fatty acids with a C14-C20 carbon chain such as 1,2- dimyristoleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine,l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dieicosenoyl-sn- glycero-3-phosphocholine; and/or 3) Natural lipids comprising a mixture of fatty acids with C12-C20 carbon chain, such as Egg PC, and Soy PC, sphingomyelin, and the like.
  • lipid bilayer for formation of a drug delivery nanocarrier (e.g., a bilayer- coated nanoparticle).
  • the drug carrier comprises bilayer comprising a lipid
  • a phospholipid e.g., a phospholipid
  • cholesterol e.g., a phospholipid
  • PEG functionalized lipid e.g., a mPEG
  • the mPEG phospholipids comprises a C14-C18 phospholipid carbon chain from, and a PEG molecular weight from 350-5000 (e.g., MPEG 5000, MPEG 3000, MPEG 2000, MPEG 1000, MPEG 750, MPEG 550, MPEG 350, and the like).
  • the mPEG phospholipid comprises DSPE-PEG5000, DSPE- PEG3000, DSPE-PEG2000, DSPE-PEG1000, DSPE-PEG750, DSPE-PEG550, or DSPE- PEG350.
  • lipid bilayer comprises an mPEG phospholipid with a phospholipid C14-C18 carbon chain, and a PEG molecular weight ranging from about 350 Da to 5000 Da. In certain embodiments the lipid bilayer comprises DPSE-PEG 2K .
  • the lipid bilayer comprises 1,2-distearoyl-sn-glycero-
  • the IDO inhibitor is conjugated to a moiety that forms a component of a vesicle structure in aqueous solution and is provided in the lipid bilayer (see, e.g., conjugated IDO inhibitors, supra.).
  • the IDO inhibitor is conjugated to a moiety such as a lipid, PHGP, vitamin E, cholesterol, and a fatty acid (see, e.g., Examples 1 and 2).
  • the IDO inhibitor is conjugated directly to the vesicle-forming moiety and in other embodiments the IDO inhibitor is conjugated to the vesicle-forming moiety via a linker (e.g., via a homo-bifunctional or hetero-bifunctional linker). In certain embodiments the linker comprises an linker.
  • the inhibitor of the IDO pathway is conjugated to a lipid, and/or to PGHP and/or to vitamin E, and/or to cholesterol (CHOL), and/or to a fatty acid (e.g., oleic acid, docosahexaenoic acid, etc.).
  • a fatty acid e.g., oleic acid, docosahexaenoic acid, etc.
  • the IDO inhibitor is conjugated to a lipid.
  • the IDO inhibitor is conjugated to a phospholipid comprising said lipid bilayer or to cholesterol comprising said lipid bilayer. In certain embodiments the IDO inhibitor is conjugated to l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine (PL). In certain embodiments the IDO inhibitor conjugated to a phospholipid has the structure of IND-PL (see, e.g., Figure 26). [0573] In certain embodiments the ratio of phospholipid: CHOL:PEG, is about phospholipid (50-90 mol%): CHOL (10-50 mol%) : PEG (1-10 mol%). In certain
  • the lipid bilayer comprises IND-PL/Chol/DSPE-PEG.
  • the bilayer comprises an IND-PL/Cholesterol/DSPE-PEG 2 K mixture at a molar ratio of 75:20:5.
  • the lipid bilayer is formulated to form a substantially uniform and intact bilayer encompassing the entire nanoparticle.
  • the lipid bilayer is formulated so that the mesoporous silica nanoparticle is colloidally stable.
  • nanomaterials are effective ICD inducers (see, e.g., Example 7).
  • these ICD-inducing nanomaterials can be administered simply as nanoparticles.
  • the nano particles can be combined with a lipid where the lipid is associated with (e.g., complexed with or conjugated to) an IDO pathway inhibitor (e.g., indoximod).
  • the lipid compires IND-PL (formula I). The lipid readily coats all or a part of the surface of the nanoparticle.
  • a nanomaterial carrier for the combined delivery of an inhibitor of an IDO pathway and an inducer of immunogenic cell death (ICD), is provided wthere the nanomaterial carrier comprises a nanomaterial that induces ICD; and a lipid or lipid formulation comprising an IDO pathway inhibitor where the lipid or lipid formulation is disposed on the surface of said nanomaterial.
  • the lipid or lipid formulation fully encapsulates the nanomaterial, while in other embodiments, the lipid or lipid formulation is disposed on a surface of the nanoparticle, but does not fully encapsulate the nanoparticle.
  • the lipid or lipid formulation can form a lipid bilayer, while more typically, the lipid or lipid formulation is not a lipid bilayer.
  • the ICD-inducing nanomaterial comprises one or more
  • ICD-inducing nanomaterials selected from the group consisting of CuO, Cu 2 0, Sb 2 0 3 , As 2 0 3 , Bi 2 0 3 , P 2 0 3 , ZnO, Ti0 2 , graphene oxide, 2D materials other than graphene or graphene oxide (e.g., graphene, graphyne, borophene, germanene, silicene, Si 2 BN, stanene, phosphorene, bismuthene, molybdenite, metals, 2D supracrystals, and the like) and other ICD-inducing nanomaterials as described herein.
  • the nanomaterial comprises copper oxide (CuO).
  • the nanomaterial comprises Sb 2 0 3 .
  • the nanomaterial comprises graphene oxide (GO).
  • the IDO pathway inhibitor associated with the lipid or lipid formulation comprises an agent selected from the group consisting of 1 -methyl -D- tryptophan (indoximod, D-1MT), L-1MT, methylthiohydantoin-dl-tryptophan (MTH-Trp, Necrostatin), ⁇ -carbolines (e.g., 3-butyl-P-carboline), naphthoquinone-based (e.g., annulin- B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S- methyl-dithiocarbamate,
  • the IDO pathway inhibitor associated with the lipid or lipid formulation comprises 1 methyl- tryptophan (1MT)).
  • the 1 methyl -tryptophan is a substantially pure "D" isomer of 1-methyl-tiyptophan (D-1MT), while in other embodiments, the 1-methyl- tryptophan is a substantially pure "L” isomer of 1 -methyl -tryptophan "L-1MT.
  • the 1-methyl-tiyptophan comprises a mixture of the D and L isomers.
  • the IDO pathway inhibitor is conjugated to a lipid or to a component of the lipid formulation.
  • the IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid.
  • the IDO inhibitor is conjugated directly to the moiety, while in other emobodiments, the IDO inhibitor is conjugated to the moiety via a linker.
  • the IDO pathway inhibitor is conjugated to PGHP, vitamin E, cholesterol (CHOL), a fatty acid, (e.g., oleic acid or docosahexaenoic acid), or to a lipid (e.g., a phospholipid).
  • the IDO pathway inhibitor is conjugated to a phospholipid.
  • Illustrative phospholipids include, but are not limited to phospholipids comprising a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains.
  • the phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC),
  • the phospholipid comprises a natural lipid selected from the group consisting of egg phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC).
  • the phospholipid comprises an unsaturated fatty acid selected from the group consisting of l,2-dimyristoleoyl-sn-glycero-3- phosphocholine, 1 ,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine, 1 ,2-dioleoyl-sn-glycero-3 - phosphocholine (DOPC), and l,2-dieicosenoyl-sn-glycero-3 -phosphocholine.
  • the phospholipid comprises l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine.
  • the IDO pathway inhibitor comprises an agent selected from the group consisting of 1-methyl-D-tiyptophan (indoximod), 1-methyl-L- tryptophan, methylthiohydantoin-dl -tryptophan, Necrostatin-1, Ebselen, Pyridoxal
  • the IDO pathway inhibitor comprises indoximod.
  • the IDO pathway inhibitor comprises substantially pure "L” isomer of 1-methyl-tiyptophan, or a substantially pure “D” isomer of 1-methyl-tiyptophan, or a racemic mixture of "D" and "L” isomers of 1 -methyl -tryptophan.
  • the 1 -methyl -tryptophan is conjugated to l-palmitoyl-2-hydroxy-5 «-glycero-3 -phosphocholine (e.g., IND-PL, Formula I).
  • Approach 2 Local treatment of a tumor or peritumor site to inhibit the IDO pathway and to induce ICD.
  • a second treatment modality involves local delivery to a tumor or peri- tumoral region, of an agent that induces ICD (e.g., doxirubicin, oxaliplatin, etc.) in combination with an inhibitor of the IDO pathway (e.g., indoximod).
  • an agent that induces ICD e.g., doxirubicin, oxaliplatin, etc.
  • IDO pathway e.g., indoximod
  • the IDO inhibitor can be complexed with or conjugated to a moiety (e.g., a lipid) that forms a vesicle (e.g., a nanovesicle).
  • a moiety e.g., a lipid
  • a vesicle e.g., a nanovesicle.
  • a method of treating a cancer in a mammal involves administering to an intra-tumoral or peritumoral site an effective amount of an inhibitor of the indoleamine 2, 3 -di oxygenase ( DO) pathway (an IDO inhibitor) in conjunction with an effective amount of an agent that induces immunogenic cell death (ICD) (an ICD-inducer).
  • the effective amount of the ICD-inducer is an amount effective to elevate calreticulin (CRT) expression and/or to elevate expression and/or release of HMGBl and/or introduce ATP release in cells of the cancer.
  • ICD inducers are well known to those of skill in the art and ICD inducers suitable for this method will readily be recognized in view of the teachings provided herein.
  • Illustrative ICD inducers include, but are not limited to chemotherapeutic agent(s) that induce ICD such as oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, oxaliplatin, paclitaxel, R2016 (a heterocyclic quinolone derivative described by Son et al. (2017) Plos One, DOI: 10.1371, which is incorporated herein by reference for the compounds described therein), and cyclophosphamide.
  • ICD inducers include oncolytic viruses (see, e.g., Angel ova et al. (2014) J. Virol, 88(10): 5263-52760.
  • One illustrative suitable oncolytic virus is an oncolytic parvovirus ⁇ e.g., H-PV).
  • the ability to induce ICD is an intrinsic property of the nanomaterial ⁇ e.g., chemical reaction of the material and/or receptor binding of the nanomaterial is not required for induction of ICD). Accordingly, in certain embodiments the tumor or peritumoral space is treated with a nanomaterial that induces ICD.
  • Such materials include, but are not limited to e.g., CuO, Cu 2 0, Sb 2 0 3 , As 2 0 3 , Bi 2 0 3 , P 2 0 3 , ZnO, Ti0 2 , graphene oxide, 2D materials other than graphene or graphene oxide ⁇ e.g., graphene, graphyne, borophene, germanene, silicene, Si 2 BN, stanene, phosphorene, bismuthene, molybdenite, metals, 2D supracrystals, etc.) and the like) ⁇ see, e.g., Example 2) nanoparticles comprising such materials. In certain embodiments the nanoparticle is entirely fabricated from said materials.
  • the nanoparticle comprises a doped material containing said materials.
  • the nanoparticle comprises a core-shell structure compmrising said ICD inducing materials. Accordingly, in certain embodiments ICD is induced by contacting the cancer cells with a nanomaterial (e.g., CuO, Sb 2 0 3 , ZnO, Ti0 2 , and graphene oxide) that induced ICD.
  • a nanomaterial e.g., CuO, Sb 2 0 3 , ZnO, Ti0 2 , and graphene oxide
  • two or more ICD inducers can be used to induce ICD via local delivery.
  • the ICD inducer comprises at least oxaliplatin, or doxirubicin e.g., as described in Examples 3 and 4.
  • the ICD inducer can be used in conjunction with an IDO inhibitor.
  • IDO inhibitors Numerous IDO inhibitors are known to those of skill in the art (see, discussion below) and the use of one or more of these IDO inhibitors is contemplated.
  • the IDO inhibitor(s) comprise a conjugated IDO inhibitor as described herein.
  • the IDO inhibitors comprise indoximod or a conjugated indoximod as described below and in Examples 1 and 2.
  • the IDO inhibitors comprise substantially pure "D" indoximod, or substantially pure “L” indoximod, or conjugated substantially pure "D” indoximod, or conjugated substantially pure "L” indoximod.
  • the ICD inducer and the inhibitor of the IDO pathway are delivered locally to a target site.
  • the ICD inducer and the inhibitor of the IDO pathway can be delivered directly to a tumor site, e.g., by injection, or through a cannula.
  • the ICD inducer and the inhibitor of the IDO pathway are delivered into a tumor mass and/or into a peritumoral site.
  • the ICD inducer and the inhibitor of the IDO pathway can be delivered as separate reagents. Alternatively, they can be delivered as a combined formulation.
  • the combined formulation comprise nanovesicles and/or lipid bilayer coated silica nanoparticles, e.g. as described herein, or suitable other dual delivery carriers that contain an IDO inhibitor plus a nanomaterial capable of inducing ICD.
  • the ICD inducer and the IDO pathway inhibitor are delivered via an implantable depot delivery system (e.g., encapsulated in a controlled release polymer, a hydrogel, and the like).
  • both the ICD inducer and the the IDO pathway inhibitor are in implantable depot delivery systems and in other embodiments only the the IDO pathway inhibitor or the ICD inducer is in an implantable depot delivery system.
  • the ICD inducer and the IDO pathway inhibitor are used in combination as a primary therapy. In certain embodiments the ICD inducer and the IDO pathway inhibitor are used as an adjunct therapy, e.g., in combination with other chemotherapeutics, and/or surgery, and/or radio therapy. In certain embodiments the ICD inducer and the the IDO pathway inhibitor are delivered to a surgical site during or after removal of a tumor mass. [0592] In view of the examples and teachings provided herein, it will be recognized that the co-delivery of an ICD inducer and the IDO pathway inhibitor will find use in the treatment of a number of cancers.
  • Illustrative cancers include, but are not limited to pancreatic ductal adenocarcinoma (PDAC), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Adrenocortical carcinoma, Kaposi sarcoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors,
  • PDAC pancreatic ductal adenocarcinoma
  • ALL acute lymphoblastic leukemia
  • craniopharyngioma ependymoma, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g.
  • bile extrahepatic
  • ductal carcinoma in situ DCIS
  • embryonal tumors endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhan
  • lymphoblastic ALL
  • AML acute myeloid
  • CLL chronic lymphocytic
  • CML chronic myelogenous
  • hairy cell lip and oral cavity cancer
  • liver cancer primary
  • lobular carcinoma in situ LCIS
  • lung cancer ⁇ e.g., childhood, non-small cell, small cell
  • lymphoma ⁇ e.g., AIDS-related, Burkitt ⁇ e.g., non-Hodgkin lymphoma
  • cutaneous T-Cell ⁇ e.g., mycosis fungoides, Sezary syndrome
  • Hodgkin non-Hodgkin
  • primary central nervous system CNS
  • macroglobulinemia Waldenstrom
  • male breast cancer malignant fibrous
  • histiocytoma of bone and osteosarcoma melanoma ⁇ e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple
  • myeloma/plasma cell neoplasm mycosis fungoides, myelodysplastic syndromes,
  • the cancer to be treated is cancer pancreatic ductal adenocarcinoma (PDAC) and in certain embodiments, the ICD inducer comprises oxaliplatin and the IDO inhibitor comprises indoximod or a conjugated indoximod as described below in in Example 1.
  • Approach 3 Vaccination to prevent or treat a cancer
  • methods for the prevention or treatment of a cancer that involve vaccinating a subject (e.g., a human, or a non-human mammal) to induce an immune response directed against one or more cancers. It was a surprising discovery that vaccination of a mammal with cancer cells in which ICD has been induced ex vivo is sufficient to generate a systemic immune response that can interfere with tumor growth at a remote site as well as allowing adoptive transfer to non-immune animals.
  • a subject e.g., a human, or a non-human mammal
  • vaccination methods can be used for the treatment of an existing cancer or prophylactically to prevent or inhibit the formation of a cancer in a subject.
  • subjects that have a family history for cancer in general or for particular cancers, and/or that have a genetic risk for a cancer e.g., mutations in BRCA1, and/or BRCA2, and/or P53
  • the vaccination is used as a primary therapy in the treatment of a cancer.
  • the vaccination is used as an adjunct therapy, e.g., in combination with surgery, and/or other chemotherapy regimen, and/or radiation therapy.
  • a method for the treatment and/or prevention of a cancer in a mammal comprising providing cancer cells in which immunogenic cell death (ICD) has been induced ex vivo, and vaccinating the mammal with these cells, where the vaccination induces an anti-cancer immunogenic response.
  • ICD immunogenic cell death
  • the cancer cells are cells derived from an existing cancer, e.g., obtained during a biopsy, or after surgical resection of a tumor mass).
  • the cancer cells are cells obtained from the subject that is to be treated and comprise an autologous transplant.
  • the cells are obtained from a different subject of the same species or can even be obtained from a different species.
  • the cancer cells are cells from a cancer cell line.
  • the cell line is an animal cell line from the same species that is to be treated. Similalry, where a human is to be treated a human cell line will typically be used. Numerous cancer cell lines are known to those of skill in the art. Illustrative, but non-limiting examples of suitable cell lines are shown in Table 1.
  • Table 1 Illustrative, but non-limiting, cell lines that can be used to produce dying cancer cells in which immunogenic cell death (ICD) has been induced.
  • ICD immunogenic cell death
  • HTB-13TM SW 1783 Human Brain Astrocytoma
  • CRL-1620TM A 172 Human Brain Glioblastoma
  • HTB-148TMH4 Human Brain Neuroglioma HTB-187TMD341 Med Human Brain, cerebellum Medulloblastoma
  • HTB-186TMDaoy Human Brain cerebellum Medulloblastoma, desmoplastic cerebellar
  • the cancer cells used in the vaccination are of the same type of cancer that is to be treated and/or prevented. It will be recognized however, that vaccination with cells of one type of cancer may generate an immune response directed against a different cancer and/or against multiple cancers. In certain embodiments the vaccination is with cells from multiple different types (e.g., 2 or more cancers, 3 or more cancers, 4 or more cancers, 5 or more cancers, 6 or more cancers, 7 or more cancers, 8 or more cancers, 9 or more cancers, 10 or more cancers, etc.) in which ICD is induced.
  • multiple different types e.g., 2 or more cancers, 3 or more cancers, 4 or more cancers, 5 or more cancers, 6 or more cancers, 7 or more cancers, 8 or more cancers, 9 or more cancers, 10 or more cancers, etc.
  • illustrative cancers to be treated or prevented include, but are not limited to pancreatic ductal adenocarcinoma (PDAC), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Adrenocortical carcinoma, Kaposi sarcoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymo
  • PDAC pancreatic
  • bile extrahepatic
  • ductal carcinoma in situ DCIS
  • embryonal tumors endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhan
  • lymphoblastic ALL
  • AML acute myeloid
  • CLL chronic lymphocytic
  • CML chronic myelogenous
  • hairy cell lip and oral cavity cancer
  • liver cancer primary
  • lobular carcinoma in situ LCIS
  • lung cancer e.g., childhood, non-small cell, small cell
  • lymphoma e.g., AIDS-related, Burkitt (e.g., non-Hodgkin lymphoma)
  • cutaneous T-Cell e.g., mycosis fungoides, Sezary syndrome
  • Hodgkin non-Hodgkin
  • primary central nervous system CNS
  • macroglobulinemia Waldenstrom
  • male breast cancer malignant fibrous
  • histiocytoma of bone and osteosarcoma melanoma (e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple
  • myeloma/plasma cell neoplasm mycosis fungoides, myelodysplastic syndromes,
  • the cells used in the vaccination include cells of one or more of these cancers.
  • Methods of inducing immunogenic cell death are well known to those of skill in the art.
  • ICD is induced by contacting the cells (e.g., primary tumor cells, cancer cell lines, etc.) with one or more chemotherapeutic agent(s) that induce ICD.
  • Such agents include, but are not limited to oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016 (a heterocyclic quinolone derivative described by Son et al. (2017) Plos One, DOL 10.1371, which is incorporated herein by reference for the compounds described therein), and cyclophosphamide.
  • the ICD chemo reagents may also include the drug derivatives, i.e. prodrugs, that are capable of releasing the abovementioned chemotherapeutics in biological environments.
  • ICD infecting the cells with an oncolytic virus.
  • oncoviruses that induce ICD include, but are not limited to Parvovirus (e.g., H-PV (see, e.g., Angelova et al. (2014) J. Virol, 88(10): 5263-5276), and the like), Adenovirus (AD) (e.g., hTERT-Ad (see, e.g., Boozari et al. (2010) Gut. 59: 1416- 1426), Ad5/3-D24-GMCSF (see, e.g., Liikanen et al. (2013) Mol. Ther.
  • Parvovirus e.g., H-PV (see, e.g., Angelova et al. (2014) J. Virol, 88(10): 5263-5276), and the like
  • AD e.g., hTERT-Ad (see, e.g., Boozari et al. (2010) Gut.
  • HSV Herpes simplex virus
  • G207 see, e.g., Toda et al. (1999) Hum. Gene. Ther. 10: 385-393
  • HSV-1716 see, e.g., Benencia et al. (2005) Mol. Ther., 12: 789-8020
  • T- VEC see, e.g., Hu et al. (2006) Clin. Cancer Res. 12: 6737-67470
  • HSV-2 ⁇ mutant see, e.g., Colunga et al.
  • Poxvirus e.g., vSP (see, e.g.,Guo et al. (2005) Cancer Res. 65: 9991-9998, vvDD (see, e.g., John et al. (2012) Cancer Res., 72: 1651-1660), Pexa-Vec (see, e.g., Heo et al. (2013) Nat. Med, 19: 329-336), and the like
  • Arbovirus see, e.g., VSV-GFP (Indiana serotype) (see, e.g., Wongthida et al. (2010) Cancer Res.
  • VSVgm-icv see, e.g., Lemay et al. (2012) Mol. Ther., 20: 1791-1799), and the like
  • Paramyxovirus e.g., MV-eGFP (Edmonston strain) (see, e.g., Donnelly et al. (2013) Gene Ther. 20: 7-15), and the like).
  • MV-eGFP Edmonston strain
  • ICD inducing ICD
  • radiation e.g., gamma radiation, UVC radiation
  • ICD induction is accomplished using any of the compounds and/or modalities described in Table 2.
  • ICD immunogenic cell death
  • UVC irradiation apoptotic ecto-HSP70; late apoptotic ⁇ -irradiation passively released HMGB 1
  • anthracyclines e.g., Daunorubicin,
  • Pre-apoptotic ecto-CRT pre-apoptotic secreted ATP; pre-apoptotic ecto-HSP70;
  • Coxsackievirus B3 secreted ATP; late apoptotic passively
  • Oncolytic parvovirus e.g., H-PV
  • the methods of inducing ICD can involve contacting the cells with materials, e.g., nanomaterials that induce ICD.
  • materials e.g., nanomaterials that induce ICD.
  • materials include, but are not limited to CuO, Sb 2 0 3 , ZnO, Ti0 2 , and graphene oxide (see, e.g., Example 3).
  • ICD is induced by contacting the cancer cells with a nanomaterial that induces ICD (e.g., CuO, Cu 2 0, Sb 2 0 3 , As 2 0 3 , Bi 2 0 3 , P 2 0 3 , ZnO, Ti0 2 , graphene oxide, 2D materials other than graphene or graphene oxide (e.g., graphene, graphyne, borophene, germanene, silicene, Si 2 BN, stanene, phosphorene, bismuthene, molybdenite, metals, 2D supracrystals, and the like)).
  • the nanomaterial comprises copper oxide.
  • the nanomaterial comprises Sb 2 0 3 .
  • the nanomaterial comprises graphene oxide (GO). CuO, Cu 2 0, Sb 2 0 3 , As 2 0 3 , Bi 2 0 3 , P 2 0 3 , ZnO, Ti0 2 , graphene oxide, and 2D materials other than graphene or graphene oxide
  • 2D materials other than graphene are known to those of skill in the art ⁇ see, e.g., Mas Balleste et al. (2011) Nanoscale, 3 : 20-30). Such materials include, but are not limited to graphene, graphyne, borophene, germanene, silicene, Si 2 BN, stanene, phosphorene, bismuthene, molybdenite, metals, 2D supracrystals, and the like.
  • 2D materials include, but are not limited to BN, MoS 2 , NbSe 2 , Bi 2 Sr 2 CaCu 2 0 x (Id.), single layers of single layers of manganese (see, e.g., Omomo et al. (2003) J. Am. Chem. Soc, 125: 3568- 3575), oxides of cobalt (see, e.g., Kim et al. (2009) Chem. Eur. J, 15: 10752-10761), tantalum (Fukuda et al. (2007) Inorg. Chem. 46: 4787-4789), ruthenium (Fukuda et al. (2010) Inorg. Chem.
  • these ICD-inducing nanomaterials exhibit a range of tunable physicochemical properties that can readily be adapted to achieve the optimal ICD-inducing catalytic outcomes.
  • these properties include, inter alia, nanosheet size, surface oxidation status, and the like
  • metal oxides these properties include, inter alia, the particle size, dissolution characteristics, zeta potential, and the like.
  • two or more agents can (e-g-, two or more of the agents or modalities described above) can be used to induce ICD.
  • ICD is characterized by elevated expression of calreticulin (CRT), and/or elevated expression and/or release of e.g., HMGB1 or ATP as compared to the same cells in which ICD is not induced.
  • CRT calreticulin
  • Illustrative, but non-limiting methods of inducing ICD in cancer cells (e.g., KPC cells) and evaluation of the ICD are described in Example 1.
  • injection may be performed by needle or pressure.
  • mucosal immunization can be performed and such modalities include, but are not limited to intraocular, intranasal and/or oral.
  • jet injectors such as Antares Pharma's MediJector
  • VISION deliver medication through high-speed, pressurized liquid penetration of the skin without a needle. These have been developed as single-use devices and multiuse systems. A high peak pressure behind the liquid is required so it can drill a hole in the skin, and then the pressure is reduced to allow the rest of the liquid to enter the skin.
  • Other transdermal approaches deliver the antigen in a solid form. These approaches have the added benefit that the therapeutic agent is more stable and therefore may not need cold storage.
  • Another illustrative, but non-limiting approach uses the pharmaceutical formulation itself to puncture the skin.
  • Glide Pharma has developed a low-velocity, spring- powered administrator that pushes a pointed rod of pharmaceutical material through the skin in a fraction of a second. This administrator enables constant, reliable delivery of a solid dosage form and could be applied to various vaccines including vaccines comprising cancer ICD-induced cancer cells as described herein.
  • the antigen e.g., ICD- induced cancer cells
  • the hydrogel is an injectible hydrogel.
  • Injectable hydrogels can be prepared using a wide range of materials. Cyto- and bio-compatibility as well as reactive chemistries are typical factors considered for selecting base materials that can be used in hydrogels for cell delivery. Material crosslinking (formation and concentration of physical or covalent linkages), biodegradability, and biochemical properties can influence the structural, mechanical, and biological properties of the hydrogels initially and over time. Hydrophilic polymers used for hydrogel construction generally can be divided into two categories: natural polymers derived from tissues or other natural sources and synthetic polymers fabricated using organic chemistry and molecular engineering principles.
  • Biocompatible natural polymers such as hyaluronic acid, chitosan, heparin, alginate, fibrin, collagen, chondroitin sulfate, and silk, mimic aspects of the native microenvironment, including its mechanical and biochemical properties for modulating cell adhesion, migration, and other functions (see, e.g., Munarin et al. (2012) J. Appl. Biomater. Funct. Mater. 10(2): e67-81). These natural polymers have been used as building blocks for injectable hydrogel formation by physical (e.g., ionic, hydrogen bonding) or covalent crosslinking (e.g., reaction of functional groups on modified polymers) (see, e.g., Kharkar et al. (2013) Chem. .Soc. Rev. 42(17): 7335-7372.
  • Synthetic polymers such as poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(N-isopropylacrylamide) (PNIPAAm), and polycaprolactone (PCL) have frequently been used for the design of injectable, cell-compatible hydrogels due to their commercial availability, low batch-to-batch variation, versatility for chemical modification, and consequently, the ease of tuning the mechanical properties of the resulting hydrogels. Since synthetic polymers lack the inherent biochemical cues for interaction with cells, In certain embodiments they can be used in combination with natural polymers or biomimetic peptides to facilitate cell adhesion, migration, and protein secretion.
  • PEG poly(ethylene glycol)
  • PVA poly(vinyl alcohol)
  • PNIPAAm poly(N-isopropylacrylamide)
  • PCL polycaprolactone
  • the cells can be delivered by use of an injectable (or implantable) cryogel.
  • Cryogels are a type of hydrogel made up of cross-linked hydrophilic polymer chains that can hold up to 99 percent water. They are created by freezing a solution of the polymer that is in the process of gelling. When thawed back again to room temperature, the substance turns into a highly interconnected pore- containing hydrogel, which is similar in composition to bodily soft tissues in terms of their water content, structure, and mechanics.
  • cyrogel is formed from methacrylated alginate (MA-alginate) as described by Bencherif et al. (2016) Nat. Comm., 6: 7556.
  • the vaccination utilizing cancer cells in which ICD has been induced is performed using one or more adjuvants to increase the subject's immune response to the vaccination.
  • adjuvants enhance and direct the adaptive immune response to vaccine antigens.
  • Adjuvants may exert their effects through different mechanisms. Some adjuvants, such as alum and emulsions ⁇ e.g., MF59®), function as delivery systems by generating depots that trap antigens at the injection site, providing slow release in order to continue the stimulation of the immune system. These adjuvants enhance the antigen persistence at the injection site and increase recruitment and activation of antigen presenting cells (APCs). Particulate adjuvants ⁇ e.g., alum) have the capability to bind antigens to form multi-molecular aggregates that encourage uptake by APCs ⁇ see, e.g., Leroux-Roels (2010) Vaccine. 288(3) :C25-3).
  • APCs antigen presenting cells
  • Some adjuvants are also capable of directing antigen presentation by the major histocompatibility complexes (MHC) ⁇ Id.).
  • Other adjuvants essentially ligands for pattern recognition receptors (PRR), act by inducing the innate immunity, predominantly targeting the APCs and consequently influencing the adaptive immune response.
  • PRR pattern recognition receptors
  • TLRs Toll-like receptors
  • NLRs NOD-like receptors
  • RIG-I- like receptors RLRs
  • CLRs C-type lectin receptors
  • transcription factors e.g., NF- ⁇ , IRF3
  • cytokines and chemokines that play a key role in the priming, expansion and polarization of the immune responses.
  • Activation of some members of the NLR family, such as NLRP3 and NLRC4 triggers the formation of a protein complex, called inflammasome, implicated in the induction of the pro-inflammatory cytokines IL- ⁇ (see, e.g., Li et al. (2008) J. Immunol. 181(1): 17- 21.) and IL-18.
  • the NLRP3 and NLRC4 inflammasomes have been involved in the innate immunity induced by certain adjuvants.
  • Alum is the most commonly used adjuvant in human vaccination. Alum provokes a strong Th2 response. Alum induces the immune response by a depot effect and activation of APCs. The NLRP3 inflammasome has been linked to the immunostimulatory properties of alum.
  • a high aspect ratio AIOOH variant of alum can be used as an adjuvant.
  • the high aspect ratio AIOOH that is 1-2 orders of magnitude better than Alum, based, inter alia, on the principle that the long aspect ratio of the material and its surface reactivity provide superior stimulation to the NRLP3 inflammasome in dendritic cells (see, e.g., Sun et al.
  • New adjuvants are being developed that are natural ligands or synthetic agonists for PRRs, either alone or with various formulations.
  • PRR activation stimulates the production of pro-inflammatory cytokines/chemokines and type I IFNs that increase the host's ability to eliminate the pathogen.
  • PAMPs pathogens associated molecular patterns
  • a number of these agonists are now in clinical or late preclinical stages of development (see, e.g., Steinhagen et al. (2011) 29(17): 3341-3355; Mbow et al. (2010) Curr. Opin. Immunol. 22(3): 411-416).
  • the immune response can be biased towards a Thl response (see, e.g., Didierlaurent et al. (2009) J. Immunol. 183(10): 6186-6197).
  • Double-stranded RNA (dsRNA), which is produced during the replication of most viruses, is a potent inducer of innate immunity.
  • Synthetic analogs of dsRNA such as poly(LC) have been utilized as adjuvants. They act through TLR 3 and RIG-I/MDA-5, inducing IL-12 and type I IFNs production, facilitating antigen cross-presentation to MHC class II molecules, and improving generation of cytotoxic T cells.
  • LPS Bacterial lipopolysaccharides
  • MPLA monophosphoryl lipid A
  • AS04 alum
  • Thl response a polarized Thl response and is approved for clinical use in Europe.
  • graphene oxide can interact with TLR4.
  • the TLR5 ligand bacterial flagellin
  • flagellin is a potent T-cell antigen and has been utilized as a vaccine adjuvant. Unlike other TLR agonists, flagellin tends to produce mixed Thl and Th2 responses rather than strongly Thl responses. Flagellin can be used as an adjuvant mixed with the antigen.
  • Imidazoquinolines e.g., imiquimod, gardiquimod, and R848 are synthetic compounds that activate TLR7/8 in multiple subsets of dendritic cells leading to the production of IFN-a and IL-12 thus promoting a Thl response.
  • the formulations and/or drug delivery nanocarriers described herein can easily include imiquimod.
  • Oligodeoxynucleotides containing specific CpG motifs are recognized by TLR9. They enhance antibody production and strongly polarize the cell responses to Thl and away from Th2 responses.
  • various a drug delivery nanocarriers described herein e.g., a bilayer-coated nanoparticle
  • can readily be modified to present CPG oligonucleotides on the surface e.g., LB-coated nanoparticles can present CPG oligo's on the lipid bilayer.
  • MDP muramyl dipeptide
  • Adjuvants may be combined to achieve a stronger effect or a more potent skewing of immune responses.
  • alum has been combined with TLR9 agonists (see, e.g., Siegrist et al. (2004) Vaccine, 23(5): 615-622).
  • TLR9 agonists see, e.g., Siegrist et al. (2004) Vaccine, 23(5): 615-622).
  • administration of other combinations such as CpG ODNs with MDP or MPLA has proven effective (see, e.g., Kim et al. (2000) Vaccine, 19: 530-537).
  • any one or more of the these adjuvants may be used to enhance response to the vaccination with cancer cells in which ICD has been induced.
  • IDO inhibitors are well-known to those of skill in the art and useful in the methods described herein. Illustrative, but non-limiting examples of IDO inhibitors are shown in Table 3 and the structures of several of these are shown in Figure 2. Table 3. Illustrative, but non-limiting IDO inhibitors.
  • IDO inhibitors include, but are not limited to the inhibitors described in U.S. Patent Publication Nos: US 2016/0362412, US 2016/0289171, US
  • the methods described herein can use one or more of these IDO inhibitors and/or any other IDO inhibitors known to those of skill in the art.
  • the one or more IDO inhibitors comprise indoximod.
  • one or more IDO inhibitors are conjugated to a moiety that forms a vesicle (e.g., a liposome) structure in aqueous solution or that can form a component of a lipid bilayer comprising a liposome.
  • a vesicle e.g., a liposome
  • the conjugated IDO inhibitors can be used directly (e.g., described in approach 2 above), provided as components in a combined formulation (e.g., in combination with an ICD inducer), and in certain embodiments, the IDO inhibitor is conjugated to a moiety that forms a component of a lipid bilayer that can be disposed on a nanoparticle, e.g., as described below and in Example 1).
  • the moiety that is conjugated to the the IDO pathway inhibitor comprises a lipid, PHGP, vitamin E, cholesterol, and/or a fatty acid.
  • the an ester bond is used to make the conjugate.
  • the H 2 group in the indoximod is protected before the conjugation reaction.
  • the -COOH in indoximod can then robustly react with the in the conjugating moiety (e.g., PHGP, Vitamin E, cholesterol, a fatty acid, a lipid, etc.).
  • the NH 2 group can be protected.
  • IDO inhibitors have other groups readily available for conjugation directly to a vesicle-forming moiety or to a linker.
  • groups include for example, H, OH, CH 2 , and the like (see, e.g., Figure 2).
  • the IDO pathway inhibitor can be conjugated to a lipid (e.g., a phospholipid), or cholesterol.
  • a lipid e.g., a phospholipid
  • the other vesicle-forming agents having conjugated IDO inhibitor(s) can also be incorporated into a lipid bilayer.
  • the inhibitor of the IDO pathway is conjugated to a phospholipid comprising a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains.
  • the phospholipid comprising a saturated fatty acid with a C14-C20 carbon chain
  • an unsaturated fatty acid with a C14-C20 carbon chain and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains.
  • phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC),
  • the phospholipid comprises a natural lipid selected from the group consisting of egg phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC).
  • the phospholipid comprises an unsaturated fatty acid selected from the group consisting of l,2-dimyristoleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoleoyl-sn- glycero-3-phosphocholine,l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2- dieicosenoyl-sn-glycero-3-phosphocholine.
  • unsaturated fatty acid selected from the group consisting of l,2-dimyristoleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoleoyl-sn- glycero-3-phosphocholine,l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2- dieicosenoyl-sn-glycero-3-phosphocholine.
  • the phospholipid comprises l-palmitoyl-2-hydroxy- sn-glycero-3-phosphocholine.
  • the lipid conjugated inhibitor of the IDO pathway comprises l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine conjugated to 1-methyl-tryptophan, and in certain embodiments the lipid conjugated inhibitor comprises IND-PL having the structure shown in Formula I:
  • the 1- methyl-tryptophan component of IND-PL can be a "D" isomer or an L isomer.
  • the IDO pathway inhibitors can be incorporated into the lipid bilayer forming the vesicle witout conjugation to a lipid bilayer component.
  • epacadostat is a potent direct IDO enzyme inhibitor with an IC50 of -125 nM in a whole blood assay (Yue et al. (2017) ACS Med. Chem. Letts. 8: 486-491).
  • the drug showed good synergy with anti-PDl antibody (nivolumab) in a phase II clinical trial in melanoma patients, the success could not be duplicated in a recent phase 3 clinical trial for the same disease.
  • Epacadostat is highly soluble in ethanol (>20 mg/mL), which allows its incorporation into a liposomal membrane through the use of the ethanol injection method ⁇ see, e.g., Pons, et al. (1993) J.
  • the ethanol injection method produces homogeneous unilamellar liposomes (Pereira et al. (2016) Int. J. Pharmaceutics, 514: 150-159).
  • water is poured into a concentrated lipid-ethanol solution (containing docetaxel and possibly IND- PL in a ratiometric designed strategy), following which ethanol is removed in an evaporator (Id.).
  • Dilution with water causes spontaneous formation of small and homogenous unilamellar liposomes from the micellar aggregate.
  • the size of the liposomes can be controlled by the ratio of ethanol to water.
  • encapsulation of, e.g., the ICD inducer in the nanoparticle and/or in the nanovesicle is optimized by using a "remote loading" strategy in which the addition of the drug ⁇ e.g., ICD-inducer such as doxorubicin) to preformed vesicles or silicasomes (LB-coated nanoparticles) which achieves high loading levels using a a pH gradient or an ion gradient capable of generating a pH gradient (see, e.g., Ogawa et al. (2009) J. Control. Pel. 1 (5) : 4- 10; Fritze et al. (2006) Biochimica et Biophys Acta. 11 '58: 1633-
  • the remote loading method involves adding a cargo-trapping reagent ⁇ e.g., protonating reagent such as TEA 8 SOS, ammonium sulfate, etc.) which can be added to the lipid biofilm prior to the sonication in the formation of silicasomes, or can be incorporated into the nanovesicle lipids prior to the formation of the nanovesicle e.g., as described in Example 2.
  • a cargo-trapping reagent e.g., protonating reagent such as TEA 8 SOS, ammonium sulfate, etc.
  • a DOX/IND nanovesicle cam be prepared as follows: 1) a total of 50 mg lipids of IND-PL plus other vesicle-forming lipids (e.g., IM)-PL/cholesterol/DSPE-PEG2K), in certain
  • embodiments at a molar ratio, 75:20:5 can be dissolved in 5 mL chloroform in a 50 mL round bottom glass flask.
  • the solvent can be evaporated under a rotatory vacuum to form a uniform thin lipid film, can be dried further under vacuum overnight.
  • the film can be hydrated with a cargo-trapping agent (e.g., with 2 mL of ammonium sulfate (123 mM) and probe sonicated, e.g., for 1 h, then subsequently extruded, e.g., 15 times, through a Mini- Extruder (Avanti Polar Lipids), using, e.g., a polycarbonate membrane with 100 nm pores (Avanti Polar Lipids) at 80 °C.
  • IND nanovesicle (IND-NV) size and morphology can be assessed by dynamic light scattering and cryoEM, respectively as desired.
  • Unincorporated cargo-trapping agent e.g., ammonium sulfate
  • Unincorporated cargo-trapping agent e.g., ammonium sulfate
  • the drug to be loaded e.g., 6.4 mg of DOX'HCl (10 mg/mL) in DI water
  • the nanovesicles can be fractionated across a PD-10 column, allowing the removal of free DOX.
  • Their size and morphology can be assessed by dynamic light scattering, cryoEM and UPLC/MS-MS, respectively.
  • FIG. 6 illustrates the synthesis of DOX-laden IND-PL coated MSNP.
  • a DOX/IND-MSNP dual-delivery carrier was designed by trapping DOX in the mesoporous interior of a -65 nm MSNP, using the lipid bilayer into which IND-PL was incorporated.
  • we used the previously described biofilm method for rapid encapsulation, by sonication (Meng et al. (2015) ACS Nano, 9(4): 540-3557; Liu et al. (2016) ACS Nano, 10: 2702-2715).
  • DOX was then remotely loaded using the protocol as previously described (Id).
  • this involves preparing the MSNPs, e.g., by a sol-gel synthesis process (see. e.g., Meng et al. (2015) ACS Nano, 9(4): 540-3557).
  • the MSNPs are then soaked in the cargo-trapping agent ⁇ e.g., ammonium sulfate) to load the agent into the pores of the MSNPs.
  • the lipid formulation that will comprise the bilayer surrounding the silicasome is prepared, e.g., as described in Example 2, where the lipid formulation incorporates the IDO inhibitor ⁇ e.g., IND-PL).
  • the cargo-trapping agent loaded MSNPs are added to the IDO-inhibitor lipid film followed by sonication ⁇ e.g., 30 min probe sonication) to provide the trapping agent ⁇ e.g., ammonium sulfate)-loaded IND-PL coated MSNP.
  • sonication e.g., 30 min probe sonication
  • the trapping agent e.g., ammonium sulfate
  • the particle suspension was passed through a PD-10 size exclusion column. Ammonium sulfate-containing IND-PL coated MSNPs were eluted from column faster than free ammonium sulfate due to its large size.
  • Remote Dox loading was accomplished by incubating 6.5-32.4 mg of DOX'HCl (10 mg/mL) in DI water with cargo- trapping agent loaded laden IND-PL coated MSNP at 65 °C for 40 min. The pure DOX/IND- MSNP was collected by centrifuging at 15,000 rpm for 15 min, three times.
  • This protocol also is illustrative and non-limiting. Using this teaching, numerous other silicasomes comprising an IDO pathway inhibitor and ICD-inducer and various lipid formulatiosn can be produced by one of skill in the art.
  • lipid conjugation technology described herein can be used to make prodrugs out of chemo agents, which can be folded into a liposome.
  • ICD chemo agents like the taxanes can be incorporated into a
  • the versatility of the liposomal platform described herein allows the encapsulation of ICD-inducing drugs such as paclitaxel, docetaxel, mitroxantrone and etoposide through the use different loading strategies that depend on the chemical structure of the drugs.
  • ICD-inducing drugs such as paclitaxel, docetaxel, mitroxantrone and etoposide
  • mitoxantrone which is a weak basic molecule with MW of 444.4, water solubility of 89 mg/mL and log P value of -3.1 (Mitoxantrone.
  • paclitaxel is moderately soluble in ethanol (1.5 mg/mL), up to ⁇ 5 wt% PTX can be loaded into the liposomal membrane by ethanol injection (Koudelka & Turanek(2012) J. Control. Release, 163 : 322-334).
  • a cargo-trapping reagent can be utilized to facilitate incorporation of a cargo ⁇ e.g., DOX) into the dual-delivery (ICD- inducer/IDO-inhibitor) LB coated MS P (ICD/IDO silicasome), and/or the dual-delivery lipid vesicles ⁇ e.g., ICD/IDO-lipid vesicles).
  • the cargo-trapping reagent can be selected to interact with a desired cargo. In some embodiments, this interaction can be an ionic or protonation reaction, although other modes of interaction are contemplated.
  • the cargo- trapping agent can have one or more ionic sites, i.e., can be mono-ionic or poly-ionic.
  • the ionic moiety can be cationic, anionic, or in some cases, the cargo-trapping agent can include both cationic and anionic moieties.
  • the ionic sites can be in equilibrium with corresponding uncharged forms; for example, an anionic carboxylate (-COO " ) can be in equilibrium with its corresponding carboxylic acid (-COOH); or in another example, an amine (-NH 2 ) can be in equilibrium with its corresponding protonated ammonium form (- H3 + ). These equilibriums are influenced by the pH of the local environment.
  • Certain ICD-inducing weak-base reagents such as doxorubicin, can be loaded using a trapping agent mediated approach for loading ⁇ see, e.g., Example 2).
  • the cargo can include one or more ionic sites.
  • the cargo-trapping agent and cargo can be selected to interact inside the dual-delivery (ICD-inducer/IDO-inhibitor) LB coated MSNP (ICD/IDO silicasome), and/or the dual- delivery lipid vesicle ⁇ e.g., ICD/IDO-lipid vesicle). This interaction can help retain the cargo within the nanoparticle until release of the cargo is desired.
  • the cargo can exist in a pH-dependent equilibrium between non-ionic and ionic forms. The non-ionic form can diffuse across the lipid bilayer and enter the vesicle or the pores of the MSNP.
  • the cargo-trapping agent e.g., a polyionic cargo-trapping agent
  • the cargo-trapping agent can interact with the ionic form of the cargo and thereby retain the cargo within the nanocarrier, e.g., within the vesicle or within the pores of the MSNP (provided the ionic forms of the cargo and cargo- trapping agent have opposite charges).
  • the interaction can be an ionic interaction, and can include formation of a precipitate. Trapping of cargo within the nanocarrier can provide higher levels of cargo loading compared to similar systems, e.g., nanocarriers that omit the cargo-trapping agent, or liposomes that do include a trapping agent.
  • Release of the cargo can be achieved by an appropriate change in pH to disrupt the interaction between the cargo and cargo-trapping agent, for example, by returning the cargo to its non-ionic state which can more readily diffuse across the lipid bilayer.
  • the cargo is irinotecan and the cargo-trapping agent is TEA 8 SOS.
  • the cargo trapping agent need not be limited to TEA 8 SOS.
  • the cargo trapping comprises small molecules like (NH 4 ) 2 S0 4 , and the like (see, e.g., Example 2).
  • Other trapping agents include, but are not limited to, ammonium salts (e.g., ammonium sulfate, ammonium sucrose octasulfate, ammonium a-cyclodextrin sulfate, ammonium ⁇ -cyclodextrin sulfate, ammonium ⁇ -cyclodextrin sulfate, ammonium phosphate, ammonium a-cyclodextrin phosphate, ammonium ⁇ -cyclodextrin phosphate, ammonium ⁇ - cyclodextrin phosphate, ammonium citrate, ammonium acetate, and the like),
  • ammonium salts e.g., ammonium sulfate, ammonium
  • trimethylammonium salts e.g., trimethylammonium sulfate, trimethylammonium sucrose octasulfate, trimethylammonium ⁇ -cyclodextrin sulfate, trimethylammonium ⁇ -cyclodextrin sulfate, trimethylammonium ⁇ -cyclodextrin sulfate, trimethylammonium phosphate, trimethylammonium ⁇ -cyclodextrin phosphate, trimethylammonium ⁇ -cyclodextrin phosphate, trimethylammonium citrate, trimethylammonium acetate, and the like), triethylammonium salts (e.g., triethylammonium sulfate, triethylammonium sucrose octasulfate, triethylammonium a-cyclodextrin sulfate, trimethylammonium ⁇ -cyclodextrin
  • transmembrane pH gradients can also be generated by acidic buffers (e.g. citrate) (Chou et al. (2003) J. Biosci. Bioengineer., 95(4): 405-408; Nichols et al.
  • the cargo-trapping reagent is particular suitable for use with a cargo that comprises an organic compound that includes at least one primary amine group, or at least one secondary amine group, or at least one tertiary amine group, or at least one quaternary amine group, or any combination thereof, capable of being protonated.
  • the general characteristics of these cargo molecules include the following chemical properties:
  • Example 2 Remote loading utilizing doxorubicin, with ammonium sulfate as a cargo trapping agent is described in Example 2. This is illustrative, but non-limiting.
  • drugs that can be imported across the lipid bilayer of these carriers. These include, but are not limited to, weak basic compounds, with medicinal chemical features. Such compounds include, but are not limited to alkaloids (e.g. irinotecan, topotecan, 10-hydroxycamptothecin, belotecan, rubitecan, vinorelbine, LAQ824, vinblastine, vincristine, homoharringtonine, trabectedin),
  • alkaloids e.g. irinotecan, topotecan, 10-hydroxycamptothecin, belotecan, rubitecan, vinorelbine, LAQ824, vinblastine, vincristine, homoharringtonine, trabectedin
  • anthracyclines e.g. doxorubicin, epirubicin, pirarubicin, daunorubicin, rubidomycin, valrubicin, amrubicin
  • alkaline anthracenediones e.g. mitoxantrone
  • alkaline alkylating agents e.g. cyclophosphamide, mechlorethamine, temozolomide
  • purine or pyrimidine derivatives e.g.

Abstract

In certain embodiments a platform technology for the facilitating immune therapy in the treatement of cancer is provided. In certain embodiments nanocarriers are provided that facilitate delivery of an IDO pathway inhibitor in conjunction with an inducer of cell death (ICD-inducer). In certain embodiments the IDO inhibitor is conjugated to a component of a lipid bilayer forming a nanovesicle. In still another embodiment, methods and compositions are provided where an ICD-inducing agent (e.g., doxirubicin, oxaliplatin, etc.) and an IDO pathway inhibitor (e.g., an IDO inhibitor -prodrug) are integrated into a nanocarrier (e.g. a lipid-bilayer (LB) -coated nanoparticle), that allows systemic delivery to orthotopic pancreatic cancer site.

Description

NANO-ENABLED IMMUNOTHERAPY IN CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of and priority to USSN 62/507,996, filed on
May 18, 2017, and to USSN 62/614,325, filed on January 5, 2018, both of which are incorporated herein by reference in their entirety for all purposes.
STATEMENT OF GOVERNMENTAL SUPPORT
[0002] This invention was made with Government support under CA198846, awarded by the National Institutes of Health. The Government has certain rights in the invention.
BACKGROUND
[0003] While treatment of patients with localized breast cancer (BC) has a survival rate of -98%, the Breast Cancer Coalition has pointed out that there is marginal improvement on mortality rate since 1975 (DeSantis et al. (2017) CA Cancer J Clin. 67: 439-448). This is particularly true for metastatic disease, where none of the current treatments {e.g., radiation, chemotherapy, and estrogen blockers) are capable of eliminating BC once metastatic spread has taken place (Howlader et al. (eds). SEER Cancer Statistics Review, 1975-2010, Nat.
Cancer Inst. Bethesda, MD, seer.cancer.gov/csr/1975_2010/, based on November 2012 SEER data submission, posted to the SEER web site, April 2013). Consequently, there is no recognized cure for metastatic disease, which is responsible for -90% of BC mortality.
[0004] Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly fatal disease with a 5-year survival outcome of less than 6% (American Cancer Society. Cancer Facts & Figures (2014). Atlanta: American Cancer Society). In spite of this dismal prognosis, the introduction of commercial nanocarriers providing paclitaxel (PTX) or irinotecan delivery has had some survival impact (Frese et al. 92012) Cancer Discov. 2(3): 260-269; Passero etal. (2016) Exp. Rev. Anticancer Therap., 16(7): 697-703). Thus, while PTX delivery by an albumin-nanocarrier can suppress the drug-resistant tumor stroma, allowing increased gemcitabine uptake, the delivery of irinotecan by a liposome can improve drug pharmacokinetics. Moreover, our own studies using mesoporous silica nanoparticles (MSNP) have shown in a robust orthotopic PDAC animal model that it is possible, in one formulation, to include smart-design features to improve irinotecan loading efficacy, carrier stability and safety over a commercial liposomal equivalent, while a second approach was to develop a ratiometric-designed drug carrier for contemporaneous and synergistic delivery of PTX and gemcitabine (Liu et al. (2016) ACS Nano, 10(2): 2702-2715; Meng et al. (2015) ACS Nano, 9(4): 3540-3557).
[0005] In spite of this bleak picture, newfound optimism has emerged with the advent of cancer immunotherapy, where the power of T-cell immunity can be invoked to treat solid cancers, including, inter alia, breast cancer, and pancreatic cancer. This is best exemplified by the use of immune checkpoint blocking antibodies, that have changed the treatment landscape for melanoma and non-small cell lung cancer (NSCLC). However, in spite of this accomplishment, the overall response rate is only 20-30%, without clear guidance to identify responders.
SUMMARY
[0006] To increase the number of responders in the treatment of cancers {e.g., breast cancer), an important strategy that we exploited is to to convert immune deplete into immune replete ("hot") tumors as a prelude to further immunomodulatory therapy. One approach was to induce immunogenic conditions at the tumor site by via induced cell death (ICD). ICD is a specialized form of tumor cell death (Kroemer et al. (2013) Ann. Rev. Immunol, 31 : 51-72) that can be triggered by specific chemotherapeutic drugs {e.g. anthracyclines, taxanes, oxaliplatin), radiation therapy, or cytotoxic viruses. ICD facilitates tumor antigen cross- presentation in. dendritic cells as a result of calreticulin (CRT) expression on the dying tumor cell surface {see, e.g., Figure 1). CRT expression provides an "eat-me" signal for dendritic cell uptake via the CD91 receptor. In addition, the stepwise release of adjuvant stimuli, including HMGB-1 (a TLR-4 ligand) and ATP (a signal that activates the RLP3
inflammasome), allows dendritic cell maturation and antigen presentation to naive T-cells at the tumor site and regional lymph nodes (Kroemer et al. (2013) Ann. Rev. Immunol, 31 : 51- 72; Kepp et al. (2014) Oncoimmunol, 3(9): e955691). This response is frequently accompanied by a reduced number of Tregs.
[0007] We proposed that ICD will allow more predictable induction of an immune replete status to allow receptor-mediated blockade or perturbation of other immune surveillance pathways to induce durable anti-tumor immunity, which also takes care of metastases. As such, ICD can strengthen the effect of immune checkpoint blocking antibodies as well as indoleamine 2, 3 -di oxygenase (IDO) inhibitors that interfere in this metabolic immune surveillance pathway. Thus, ICD provides a deliberate means of initiating and immune "hot" start for subsequent response boosting by metabolic and immune checkpoint inhibitors.
[0008] In addition to reversal of immune suppression by receptor blocking antibodies to CTLA-4, PD-1 and PD-L1, the IDO pathway is a relevant metabolic immune checkpoint pathway in breast cancer (and other cancers) because of its overexpression at the tumor site. IDO-1 is the first and rate-limiting enzymatic step in the catabolism of tryptophan in the kynurenine pathway, and exerts potent immunosuppressive effects as a result of the metabolic disturbance of the amino acid ratios (see, e.g., Prendergast et al. (2017) Cane. Res., 77(24): 6795-6811; Lob et al. (2009) Nat. Rev. Cancer, 9: 445-452). This allows the IDO effector pathway to control the activity of the mTOR pathway (T-cell activation); activation of the aryl hydrocarbon receptor (AhR) pathway; activation of GCN2 (general control nondereressible), a serine/threonine-protein kinase that senses amino acid deficiency; and development of Tregs. As a result, IDO exerts strong immunosuppressive effects in the TME and regional lymph nodes, culminating in T-cell anergy, decreased cytotoxic killing, and increased accumulation of Tregs at the tumor site (Prendergast et al. (2014) Cancer Immunol. Immunother. 63 : 721-735; Lob et al. (2009) Nat. Rev. Cancer, 9: 445-452). The increased expression of IDO is closely associated with the clinical stage and lymph node metastases in patients with breast cancer.
[0009] While a number of small molecule IDO pathway inhibitors have emerged, one of the best studied examples is 1-methyl-tiyptophan, a.k.a. indoximod (IND). Although IND has been shown to improve the impact of paclitaxel in a mouse BC model, its modest impact as an adjuvant in human cancer studies has raised concerns about its clinical efficacy. Our own animal studies have demonstrated that the water insolubility of IND contributes to an unfavorable PK, short, circulatory half-life and inadequate tumor retention to effectively interfere in in the activity of IDO, which is overexpressed at the tumor site. This served as the impetus to design nanocarriers into which IDO pathway inhibitors (e.g., IND) could be co-delivered with ICD inducers (e.g., doxirubicin).
[0010] In one illustrative, but non-limiting embodiment, this goal was accomplished by synthesizing an IDO pathway inhibitor prodrug where the IDO inhibitor (e.g., indoximod) was conjugated to a moietiy (e.g., a phosopholipid) that can be assembled into a lipid bilayer which can in turn be incorporated into a drug delivlery vehicle (e.g3 a liposome). In one illustrative embodiment, this was accomplished by synthesizing IND as a phospholipid- conjugated prodrug that self-assembles into a nanovesicle (liposome). Not only did the IND encapsulation exert a major effect on tumor IND levels, but also provided the lipid bilayer backbone of the carrier into which an ICD inducer (e.g., DOX ) could be loaded by remote import.
[0011] Our data show that a doxorubicin (DOX) encapsulating nanocarrier provides a more potent ICD stimulus than the free drug, and can do so synergistically with a small molecule inhibitor (indoximod) of the IDO-1 pathway. The nanocarrier is capable of facilitating this task, in part, by improving the PK of DOX and indoximod (IND) at the tumor site. This provides us with a next generation nanocarrier providing an ICD stimulus and an IDO inhibitor as a promising synergistic immunotherapy platform for BC, including triple negative BC (TNBC) (most responsive to immune checkpoint inhibitors) as well as ER- positive tumors (numerically the largest BC subtype responsible for mortality) and other cancers (e.g., PDAC, and the like).
[0012] Moreover, we demonstrate the feasibility of achieving tumor regression or eradication of breast cancer tumors, and Kras-induced PDAC tumors, grown subcutaneously or orthotopically implanted, by using the nanocarriers in three different approaches: (1) systemic administration; (2) local tumor injection; and (3) vaccination. Our data show that the synergy between ICD and interference in the IDO pathway boosts innate and adaptive immunity and leads to effective killing of pancreatic cancer cells by CD8+ cytotoxic T cells at the tumor site, as well as interfering in metastatic spread. The cytotoxic response is accompanied by disappearance of Tregs at the tumor site, in addition to evidence of boosting innate immunity through increased CRT and toll-like receptor 4 (TLR4) expressions. Similar immunophenotypic changes occur in breast cancer. The systemic immune response could also be adoptively transferred to non-immune animals.
[0013] Accordingly, in certain embodiments, compositions and methods are provided for systemic and/or for local (peri- or intratumoral) delivery of one or more ICD-inducing agents (e.g., doxirubicin, oxaliplatin, etc.) in conjunction with delivery of an inhibitor of the IDO pathway (e.g., indoximod). In certain embodiments the inhibitor of the IDO inhibitor is conjugated to a nanovesicle-forming moiety (e.g., containing a phospholipid bilayer). In still another embodiment, methods and compositions are provided where an ICD-inducing agent (e.g., oxaliplatin, doxirubicin, etc.) and an IDO inhibiting agent (e.g., an IDO inhibitor - prodrug) are integrated into a nanocarrier, that allows systemic delivery to a cancer site. Additioanlly, in certain embodiments, compositions and methods are provided for the treatment or prevention of a cancer via vaccination (e.g., subcutaneous vaccination), utilizing certain cancer cells (e.g., drug-treated cancer cells) in which ICD has been induced ex vivo. It was discovered that vaccination with dying cells initiates a systemic immune response that prevents tumor re-growth.
[0014] Various embodiments contemplated herein may include, but need not be limited to, one or more of the following:
[0015] Embodiment 1 : A nanovesicle drug carrier for the combined delivery of an
IDO pathway inhibitor and an inducer of immunogenic cell death (ICD), said nanovesicle drug carrier comprising:
[0016] a lipid vesicle wherein said lipid vesicle comprises a lipid effective to form a vesicle comprising a lipid bilayer in an aqueous solution, and the lipid bilayer is associated with an inhibitor of the indoleamine 2,3 -di oxygenase (IDO) pathway (IDO pathway inhibitor); and
[0017] a cargo within said vesicle where said cargo comprises an agent that induces immunogenic cell death (ICD) (ICD-inducer). In certain embodiments the IDO pathway inhibitor can be conjugated to a component tof the lipid bilayer forming the vesicle. In certain embodiments the IDO pathway inhibitor is incorporated into the lipid bilayer (e.g., via alcohol injection, or other methods).
[0018] Embodiment 2: The nanovesicle drug carrier of embodiment 1, wherein the
IDO pathway inhibitor and the ICD inducer are synergistic in their activity against a cancer. [0019] Embodiment 3 : The nanovesicle drug carrier according to any one of embodiments 1-2, wherein said drug carrier, when administered systemically, delivers an amount of an ICD inducer effective to induce or to facilitate induction of immunogenic cell death of a cancer cell at a tumor site.
[0020] Embodiment 4: The nanovesicle drug carrier according to any one of embodiments 1-3, wherein said drug carrier, when administered systemically, delivers an amount of an IDO pathway inhibitor to partially or fully inhibit the IDO enzyme or IDO pathway at a cancer site.
[0021] Embodiment 5: The nanovesicle drug carrier according to any one of embodiments 1-4, wherein said IDO pathway inhibitor comprises an inhibitor of the IDO enzyme. [0022] Embodiment 6: The nanovesicle drug carrier according to any one of embodiments 1-5, wherein said IDO pathway inhibitor comprises an inhibitor of the IDO pathway downstream from said IDO enzyme.
[0023] Embodiment 7: The nanovesicle drug carrier according to any one of embodiments 1-6, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l-methyl-tiyptophan (L-1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tiyptophan (L-1MT),
methylthiohydantoin-dl -tryptophan (MTH-Trp, Necrostatin), β-carbolines (e.g., 3-butyl-P- carboline), Naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S- methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl- brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol-3-yl)ethyl]- S[(naphth-2-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-3-yl)methyl]- dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-4-yl)methyl]-dithiocarbamate, 5-bromo- brassinin, Phenylimidazole-based IDO inhibitors (e.g., 4-phenylimidazole), Exiguamine A, imidodicarbonimidic diamide,N-methyl-N'-9-phenanthrenyl-monohydrochloride
(NSC401366), INCB024360 (epacadostat), l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDO 1 -derived peptide, NLG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole.
[0024] Embodiment 8: The nanovesicle drug carrier of embodiment 7, wherein said
IDO pathway inhibitor comprises 1 -methyl -tryptophan.
[0025] Embodiment 9: The nanovesicle drug carrier of embodiment 8, wherein said
IDO pathway inhibitor comprises a "D" enantiomer of 1 -methyl -tryptophan (indoximod, 1- MT).
[0026] Embodiment 10: The nanovesicle drug carrier of embodiment 8, wherein said
IDO pathway inhibitor comprises an "L" enantiomer of 1-methyl-tiyptophan (L-MT).
[0027] Embodiment 1 1 : The nanovesicle drug carrier according to any one of embodiments 1-10, wherein said IDO pathway inhibitor, is disposed in a lipid comprising said vesicle and/or conjugated to a lipid comprising said vesicle. [0028] Embodiment 12: The nanovesicle drug carrier according to any one of embodiments 1-10, wherein said vesicle comprises a phospholipid and/or a phospholipid prodrug.
[0029] Embodiment 13 : The nanovesicle drug carrier of embodiment 12, wherein said vesicle comprises a phospholipid, and cholesterol (CHOL).
[0030] Embodiment 14: The nanovesicle drug carrier according to any one of embodiments 12-13, wherein said phospholipid comprises a saturated fatty acid with a C14- C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains. [0031] Embodiment 15: The nanovesicle drug carrier of embodiment 14, wherein said phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC),
distearoylphosphatidylcholine (DSPC), and diactylphosphatidylcholine (DAPC).
[0032] Embodiment 16: The nanovesicle drug carrier of embodiment 14, wherein said phospholipid comprises a natural lipid selected from the group consisting of egg phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC).
[0033] Embodiment 17: The nanovesicle drug carrier of embodiment 14, wherein said phospholipid comprises an unsaturated fatty acid selected from the group consisting of l,2-dimyristoleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine,l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dieicosenoyl-sn- glycero-3 -phosphocholine.
[0034] Embodiment 18: The nanovesicle drug carrier according to any one of embodiments 12-17, wherein said vesicle comprises an mPEG phospholipid with a phospholipid C14-C18 carbon chain, and a PEG molecular weight ranging from about 350 Da to 5000 Da.
[0035] Embodiment 19: The nanovesicle drug carrier of embodiment 18, wherein said vesicle comprises l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (DSPE-PEG).
[0036] Embodiment 20: The nanovesicle drug carrier of embodiment 19, wherein said vesicle comprises DPSE-PEG2K. [0037] Embodiment 21 : The nanovesicle drug carrier according to any one of embodiments 1-20, wherein said IDO pathway inhibitor is conjugated to a component of said vesicle.
[0038] Embodiment 22: The nanovesicle drug carrier of embodiment 21, wherein said IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid.
[0039] Embodiment 23 : The nanovesicle drug carrier according to any one of embodiments 21-22, wherein said IDO pathway inhibitor is conjugated directly to said moiety. [0040] Embodiment 24: The nanovesicle drug carrier according to any one of embodiments 21-22, wherein said IDO pathway inhibitor is conjugated to said moiety via a linker.
[0041] Embodiment 25: The nanovesicle drug carrier according to any one of embodiments 21-22, wherein said IDO pathway inhibitor is conjugated to PGHP. [0042] Embodiment 26: The nanovesicle drug carrier according to any one of embodiments 21-24, wherein said IDO pathway inhibitor is conjugated to vitamin E.
[0043] Embodiment 27: The nanovesicle drug carrier according to any one of embodiments 21-24, wherein said IDO pathway inhibitor is conjugated to cholesterol
(CHOL), or squalene. [0044] Embodiment 28: The nanovesicle drug carrier according to any one of embodiments 21-24, wherein said IDO pathway inhibitor is conjugated to a fatty acid.
[0045] Embodiment 29: The nanovesicle drug carrier of embodiment 28, wherein said IDO pathway is conjugated to oleic acid or docosahexaenoic acid.
[0046] Embodiment 30: The nanovesicle drug carrier of embodiment 28, wherein said IDO pathway is conjugated to oleic acid or docosahexaenoic acid via an
Figure imgf000010_0001
OH linker.
[0047] Embodiment 31 : The nanovesicle drug carrier according to any one of embodiments 21-24, wherein said IDO pathway inhibitor is conjugated to a lipid. [0048] Embodiment 32: The nanovesicle drug carrier of embodiment 31, wherein said IDO pathway inhibitor is conjugated to a phospholipid comprising said lipid vesicle, said phospholipid thereby forming a phospholipid prodrug.
[0049] Embodiment 33 : The nanovesicle drug carrier of embodiment 32, wherein said phospholipid prodrug comprises l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PL).
[0050] Embodiment 34: The nanovesicle drug carrier of embodiment 33, wherein said hospholipid prodrug comprises the structure:
Figure imgf000011_0001
[0051] Embodiment 35: The nanovesicle drug carrier of embodiment 34, wherein the
1 -methyl -tryptophan component of said conjugated IDO pathway inhibitor comprises a "D" isom r of 1 methyl-tyrptophan (indoximod) characterized by the formula:
Figure imgf000011_0002
3 O-lsomer
[0052] Embodiment 36: The nanovesicle drug carrier of embodiment 34, wherein the 1 -methyl -tryptophan component of said conjugated IDO pathway inhibitor comprises an "L" isomer of 1 methyl-tyrptophan (L-1MT) characterized by the formula:
Figure imgf000011_0003
[0053] Embodiment 37: The nanovesicle drug carrier of embodiment 34, wherein the
1 -methyl -tryptophan component of said conjugated IDO pathway inhibitor comprises a mixture of "D" and "L" isomers of 1 -methyl -tryptophan.
[0054] Embodiment 38: The nanovesicle drug carrier according to any one of embodiments 34-37, wherein said vesicle comprises IND-PL/Chol/DSPE-PEG.
[0055] Embodiment 39: The nanovesicle drug carrier of embodiment 38, wherein said vesicle comprises about 75% IND-PL, about 20% cholesterol, and about 5% DSPE- PEG2K.
[0056] Embodiment 40: The nanovesicle drug carrier according to any one of embodiments 1-39, wherein said ICD inducer comprises a chemotherapeutic agent selected from the group consisting of doxorubicin, oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
[0057] Embodiment 41 : The nanovesicle drug carrier of embodiment 40, wherein said ICD inducer comprises doxorubicin.
[0058] Embodiment 42: The nanovesicle drug carrier drug carrier according to any one of embodiments 1-41, wherein said carrier is colloidally stable.
[0059] Embodiment 43 : The nanovesicle drug carrier according to any one of embodiments 1-42, wherein when the cargo in the nanocarrier is a weak base, and said carrier comprises a cargo-trapping agent.
[0060] Embodiment 44: The nanovesicle drug carrier of embodiment 43, wherein said cargo trapping agent before reaction with the cargo drug loaded in the vesicle, is selected from the group consisting of triethylammonium sucrose octasulfate (TEA8SOS), ( EL^SC^, an ammonium salt, a trimethylammonium salt, and a triethylammonium salt. [0061] Embodiment 45: The nanovesicle drug carrier according to any one of embodiments 43-44, wherein said cargo-trapping agent before reaction with said drug is ammonium sulfate.
[0062] Embodiment 46: The nanovesicle drug carrier according to any one of embodiments 1-45, wherein said drug carrier is conjugated to a moiety selected from the group consisting of a targeting moiety, a fusogenic peptide, and a transport peptide. [0063] Embodiment 47: The nanovesicle drug carrier of embodiment 46, wherein said drug carrier is conjugated to a peptide that binds a receptor on a cancer cell or tumor blood vessel.
[0064] Embodiment 48: The nanovesicle drug carrier of embodiment 47, wherein said drug carrier is conjugated to an iRGD peptide.
[0065] Embodiment 49: The nanovesicle drug carrier of embodiment 47, wherein said drug carrier is conjugated to a targeting peptide shown in Table 5.
[0066] Embodiment 50: The nanovesicle drug carrier according to any one of embodiments 46-49, wherein said drug carrier is conjugated to transferrin, and/or ApoE, and/or folate.
[0067] Embodiment 51 : The nanovesicle drug carrier according to any one of embodiments 46-50, wherein said drug carrier is conjugated to a targeting moiety that comprises an antibody that binds to a cancer marker.
[0068] Embodiment 52: The nanovesicle drug carrier of embodiment 51, wherein said drug carrier is conjugated to a targeting moiety that comprises an antibody that binds a cancer marker shown in Table 4.
[0069] Embodiment 53 : The nanovesicle drug carrier according to any one of embodiments 51-52, wherein said antibody comprises a full-length antibody (intact immunoglobulin). [0070] Embodiment 54: The nanovesicle drug carrier according to any one of embodiments 51-52, wherein said antibody comprises an antibody fragment.
[0071] Embodiment 55: The nanovesicle drug carrier according to any one of embodiments 51-52, wherein said antibody is selected from the group consisting of an intact immunoglobulin, an F(ab)'2, a Fab, a single chain antibody, a diabody, an affibody, a unibody, and a nanobody.
[0072] Embodiment 56: The nanovesicle drug carrier according to any one of embodiments 1-55, wherein said drug carriers in suspension are stable for at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months when stored at 4°C. [0073] Embodiment 57: The nanovesicle drug carrier according to any one of embodiments 1-56, wherein said nanoparticle drug carrier forms a stable suspension on rehydration after lyophilization.
[0074] Embodiment 58: The nanovesicle drug carrier according to any one of embodiments 1-57, wherein said nanoparticle drug carriers, show reduced drug toxicity as compared to free drug.
[0075] Embodiment 59: The nanovesicle drug carrier according to any one of embodiments 1-58, wherein said nanoparticle drug carrier has colloidal stability in physiological fluids with pH 7.4 and remains monodisperse to allow systemic biodistribution and is capable of entering a disease site by vascular leakage (EPR effect) or transcytosis.
[0076] Embodiment 60: The nanovesicle drug carrier according to any one of embodiments 1-59, wherein said cargo within said vesicle comprises an agent that induces immunogenic cell death (ICD) selected from the group consisting of oxaliplatin,
anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, cyclophosphamide, and a bioreactive nanomaterial that induces ICD.
[0077] Embodiment 61 : The nanovesicle drug carrier of embodiment 60, wherein said cargo comprises oxaliplatin.
[0078] Embodiment 62: The nanovesicle drug carrier of embodiment 60, wherein said cargo comprises doxorubicin.
[0079] Embodiment 63 : The nanovesicle drug carrier of embodiment 60, wherein said cargo comprises a bioreactive nanomaterial that induces ICD and/or innate immune activation.
[0080] Embodiment 64: The nanovesicle drug carrier of embodiment 63, wherein said cargo comprises a nanomaterial that induces ICD where said nanomaterial is selected from the group consisting of CuO, Cu20, Sb203, As203, Bi203, P203, ZnO, Ti02, graphene oxide, and bioreactive 2D materials other than graphene or graphene oxide.
[0081] Embodiment 65 : The nanovesicle drug carrier of embodiment 64, wherein said nanomaterial comprises copper oxide (e.g. CuO). [0082] Embodiment 66: The nanovesicle drug carrier of embodiment 64, wherein said nanomaterial comprises Sb203. [0083] Embodiment 67: A method of treating a cancer, said method comprising:
[0084] administering to a subject in need thereof an effective amount of a nanovesicle drug carrier according to any one of embodiments 1-66.
[0085] Embodiment 68: The method of embodiment 67, wherein the ICD inducer and the IDO inhibitor are synergistic in their activity against said cancer.
[0086] Embodiment 69: The method according to any one of embodiments 67-68, wherein said ICD-inducer is in an amount effective to elevate calreticulin (CRT) expression in cells of said cancer.
[0087] Embodiment 70: The method according to any one of embodiments 67-69, wherein said said ICD-inducer is in an amount effective to elevate expression and/or release of HMGB 1 and/or induction of ATP release.
[0088] Embodiment 71 : The method according to any one of embodiments 67-70, wherein said administering to a subject in need thereof an effective amount of a nanovesicle drug carrier comprises a primary therapy in a chemotherapeutic regimen. [0089] Embodiment 72: The method according to any one of embodiments 67-70, wherein said administering to a subject in need thereof an effective amount of a nanovesicle drug carrier comprises an adjunct therapy in a treatment regime that additionally comprises chemotherapy using another chemotherapeutic agent, and/or surgical resection of a tumor mass, and/or radiotherapy. [0090] Embodiment 73 : The method according to any one of embodiments 67-72, wherein said nanoparticle drug carrier and/or said pharmaceutical formulation is a component in a multi-drug chemotherapeutic regimen.
[0091] Embodiment 74: The method according to any one of embodiments 67-73, wherein said cancer is pancreatic ductal adenocarcinoma (PDAC). [0092] Embodiment 75 : The method according to any one of embodiments 67-73, wherein said cancer is a cancer selected from the group consisting of breast cancer, lung cancer, melanoma, pancreas cancer, liver cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi sarcoma, lymphoma), anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic
myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer,
esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kaposi sarcoma, kidney cancer (e.g., renal cell, Wilm's tumor, and other kidney tumors), langerhans cell histiocytosis, laryngeal cancer, leukemia, acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myelogenous (CML), hairy cell, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., childhood, non-small cell, small cell), lymphoma (e.g., AIDS-related, Burkitt (e.g., non-Hodgkin lymphoma), cutaneous T-Cell (e.g., mycosis fungoides, Sezary syndrome), Hodgkin, non-Hodgkin, primary central nervous system (CNS)), macroglobulinemia, Waldenstrom, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma (e.g., childhood, intraocular
(eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple
myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes,
Myelogenous Leukemia, Chronic (CML), multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer , pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Ewing, Kaposi, osteosarcoma, rhadomyosarcoma, soft tissue, uterine), Sezary syndrome, skin cancer (e.g., melanoma, merkel cell carcinoma, basal cell carcinoma, nonmelanoma), small intestine cancer, squamous cell carcinoma, squamous neck cancer with occult primary, stomach (gastric) cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom
macroglobulinemia, and Wilm's tumor. [0093] Embodiment 76: The method according to any one of embodiments 67-75, wherein said administration is via a route selected from the group consisting of intravenous administration, intraarterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation (including intranasal and intratracheal delivery, intracranial administration via a cannula, and subcutaneous or intramuscular depot deposition.
[0094] Embodiment 77: The method according to any one of embodiments 67-75, wherein said administration comprises systemic administration via injection or cannula.
[0095] Embodiment 78: The method according to any one of embodiments 67-75, wherein said administration is administration to an intra-tumoral or peri-tumoral site. [0096] Embodiment 79: The method according to any one of embodiments 67-78, wherein said mammal is a human.
[0097] Embodiment 80: The method according to any one of embodiments 67-78, wherein said mammal is a non-human mammal.
[0098] Embodiment 81 : The method according to any one of embodiments 67-80, wherein said cancer comprises PDAC, and said IDO pathway inhibitor comprises an agent selected from the group consisting of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l- methyl-tryptophan (L-1MT), and a mixture of D-1MT and L-1MT, epacadostat.
[0099] Embodiment 82: The method of embodiment 81, wherein said inhibitor of an
IDO pathway comprises D-l -methyl-try ptophan (indoximod, D-1MT). [0100] Embodiment 83 : The method of embodiment 81, wherein said inhibitor of an
IDO pathway comprises L-l -methyl -try ptophan (L-1MT). [0101] Embodiment 84: The method of embodiment 81, wherein said inhibitor of an
IDO pathway comprises a mixture of D-1MT and L-1MT.
[0102] Embodiment 85 : The method according to any one of embodiments 67-84, wherein said cargo within said vesicle comprises one or more drugs selected from the group consisting of doxorubicin, paclitaxel, docetaxel, cyclophosphamide, mitroxantrone, etoposide, and bortezomib.
[0103] Embodiment 86: The method of embodiment 85, wherein said cargo within said vesicle comprises doxorubicin.
[0104] Embodiment 87: The method according to any one of embodiments 85-86, wherein said cancer is a breast cancer.
[0105] Embodiment 88: The method according to any one of embodiments 67-84, wherein said cargo within said vesicle comprises one or more drugs selected from the group consisting of oxaliplatin, gemcitabine, taxanes (e.g., paclitaxel and docetaxel), doxorubicin, and etoposide. [0106] Embodiment 89: The method of embodiment 88, wherein said cargo within said vesicle comprises oxaliplatin.
[0107] Embodiment 90: The method according to any one of embodiments 88-89, wherein said cancer comprise pancreatic cancer.
[0108] Embodiment 91 : The method according to any one of embodiments 67-84, wherein said cargo within said vesicle comprises one or more drugs selected from the group consisting of a taxane (e.g., paclitaxel and docetaxel), gemcitabine, a Pt-based drug (e.g., cisplatin, oxaliplatin, carboplatin), cyclophosphamide, oxaliplatin plus cyclophosphamide, epirubicin (anthracycline), and etoposide.
[0109] Embodiment 92: The method of embodiment 91, wherein said cancer comprises lung cancer.
[0110] Embodiment 93 : The method according to any one of embodiments 67-92, wherein said nanovesicle drug carrier is administered in conjunction with administration of an immune checkpoint inhibitor.
[0111] Embodiment 94: The method of embodiment 93, wherein said immune checkpoint inhibitor comprises an inhibitor of PD-1, PD-L1, PD-L2, PD-L3, PD-L4, CTLA- 4, LAG3, B7-H3, B7-H4, KIR and/or TIM3. [0112] Embodiment 95: The method of embodiment 94, wherein said checkpoint inhibitor comprises an antibody that inhibits a moiety selected from the group consisting of PD-1, PD-L1, and CTLA4.
[0113] Embodiment 96: The method of embodiment 95, wherein said antibody comprises an antibody that inhibits PD-1.
[0114] Embodiment 97: The method of embodiment 96, wherein said antibody comprises Pembrolizumab (Keytruda), or Nivolumab (Opdivo).
[0115] Embodiment 98: The method of embodiment 95, wherein said antibody comprises an antibody that inhibits PD-L1. [0116] Embodiment 99: The method of embodiment 98, wherein said antibody comprises Atezolizumab (Tecentriq), Avelumab (Bavencio), or Durvalumab (Imfinzi).
[0117] Embodiment 100: The method of embodiment 95, wherein said antibody comprises an antibody that inhibits CTLA-4.
[0118] Embodiment 101 : The method of embodiment 100, wherein said antibody comprises Ipilimumab (Yervoy).
[0119] Embodiment 102: The method according to any one of embodiments 93-101, wherein the activity of said nanovesicle drug carrier and said immune checkpoint inhibitor is synergistic.
[0120] Embodiment 103 : A composition comprising an IDO pathway inhibitor conjugated to a moiety that forms a nanovesicle in aqueous solution.
[0121] Embodiment 104: The composition of embodiment 103, wherein said IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid.
[0122] Embodiment 105: The composition according to any one of embodiments 103-104, wherein IDO pathway inhibitor is conjugated directly to said moiety.
[0123] Embodiment 106: The composition according to any one of embodiments
103-104, wherein IDO pathway inhibitor is conjugated to said moiety via a linker.
[0124] Embodiment 107: The composition of embodiment 106, wherein said linker comprises squalene. [0125] Embodiment 108: The composition according to any one of embodiments
104-107, wherein said IDO pathway inhibitor is conjugated to PGHP.
[0126] Embodiment 109: The composition according to any one of embodiments
104-107, wherein said IDO pathway inhibitor is conjugated to vitamin E. [0127] Embodiment 110: The composition according to any one of embodiments
104-107, wherein said IDO pathway inhibitor is conjugated to cholesterol (CHOL).
[0128] Embodiment 111 : The composition according to any one of embodiments
104-107, wherein said IDO pathway inhibitor is conjugated to a fatty acid.
[0129] Embodiment 112: The composition of embodiment 111, wherein said IDO pathway inhibitor is conjugated to oleic acid or docosahexaenoic acid.
[0130] Embodiment 113 : The composition of embodiment 112, wherein said IDO pathway inhibitor is conjugated to oleic acid or docosahexaenoic acid via an HO-(CH2)n=2.5- OH linker.
[0131] Embodiment 114: The composition according to any one of embodiments 104-107, wherein said IDO pathway inhibitor is conjugated to a lipid.
[0132] Embodiment 115: The composition of embodiment 114, wherein said IDO pathway inhibitor is conjugated to a phospholipid.
[0133] Embodiment 116: The composition of embodiment 115, wherein said IDO pathway inhibitor is conjugated to a phospholipid comprising a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains.
[0134] Embodiment 117: The composition of embodiment 116, wherein said phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC),
distearoylphosphatidylcholine (DSPC), and diactylphosphatidylcholine (DAPC).
[0135] Embodiment 118: The composition of embodiment 116, wherein said phospholipid comprises a natural lipid selected from the group consisting of egg
phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC).
[0136] Embodiment 119: The composition of embodiment 116, wherein said phospholipid comprises an unsaturated fatty acid selected from the group consisting of 1,2- dimyristoleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine, l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dieicosenoyl-sn- glycero-3 -phosphocholine.
[0137] Embodiment 120: The composition of embodiment 1 16, wherein said phospholipid comprises l-palmitoy l -2-hydroxy-sn-glycero-3 -phosphocholine.
[0138] Embodiment 121 : The composition according to any one of embodiments
103-120, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of D-l-methyl-tryptophan (indoximod, D-1MT), L-l-methyl-tryptophan (L-1MT), a mixture of D-1MT and L-1MT, 1 -methyl-L-tryptophan (L- 1MT), methylthiohydantoin-dl- tryptophan (MTH-Trp, Necrostatin), β-carbolines (e.g., 3-butyl-P-carboline),
Naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol- 3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S-methyl- dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl-brassinin, N- [2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S[(naphth-2- yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-3-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-4-yl)methyl]-dithiocarbamate, 5-bromo-brassinin,
Phenylimidazole-based IDO inhibitors (e.g., 4-phenylimidazole), Exiguamine A, imidodicarbonimidic diamide,N-methyl-N'-9-phenanthrenyl-monohydrochloride
(NSC401366), INCB024360 (Epacadostat), l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDO l -derived peptide, NLG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole.
[0139] Embodiment 122: The composition according to any one of embodiments
103-120, wherein said IDO pathway inhibitor comprises 1-methyl-tiyptophan. [0140] Embodiment 123 : The composition of embodiment 122, wherein said IDO pathway inhibitor comprises a D isomer of 1-methyl-tiyptophpan.
[0141] Embodiment 124: The composition of embodiment 122, wherein said IDO pathway inhibitor comprises an L isomer of 1-methyl-tiyptophpan.
[0142] Embodiment 125 : The composition of embodiment 122, wherein said IDO pathway inhibitor comprises a mixture of D and L isomers of 1-methyl-tiyptophpan. [0143] Embodiment 126: The composition of embodiment 122, wherein the lipid conjugated IDO pathway inhibitor comprises l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine.
[0144] Embodiment 127: The composition of embodiment 126, wherein the lipid conju ated IDO pathway inhibitor comprises a compound having the structure:
Figure imgf000022_0001
[0145] Embodiment 128: The composition of embodiment 127, wherein the 1- methyl-tryptophan component of said conjugated IDO pathway inhibitor comprises a "D" isomer of 1 meth l-tyrptophan (indoximod) characterized by the formula:
Figure imgf000022_0002
[0146] Embodiment 129: The composition of embodiment 127, wherein the 1- methyl-tryptophan component of said conjugated IDO pathway inhibitor comprises an "L" isomer of 1 methyl-tyrptophan (L-1MT) characterized by the formula:
Figure imgf000022_0003
OHg i- s»mer [0147] Embodiment 130: The composition of embodiment 127, wherein the 1- methyl-tryptophan component of said conjugated IDO pathway inhibitor comprises a mixture of "D" and "L" isomers of 1 -methyl -tryptophan. [0148] Embodiment 131 : The composition according to any one of embodiments
103-130, wherein the lipid-conjugated IDO pathway inhibitor forms a component of a vesicle.
[0149] Embodiment 132: A nanoparticle drug carrier for the combined delivery of an IDO pathway inhibitor and an inducer of immunogenic cell death (ICD), said nanoparticle drug carrier comprising:
[0150] a mesoporous silica nanoparticle having a surface and defining a plurality of pores that are suitable to receive molecules therein;
[0151] a lipid bilayer coating the surface (e.g., encapsulating the nanoparticle and sealing the plurality of pores);
[0152] a first cargo comprising an inhibitor of the indoleamine 2,3- dioxygenase (IDO) pathway (IDO pathway inhibitor); and
[0153] a second cargo comprising an agent that induces immunogenic cell death (ICD) (ICD-inducer);
[0154] wherein the lipid bilayer is substantially continuous and encapsulates said nanoparticle stably sealing the plurality of pores.
[0155] Embodiment 133 : The nanoparticle drug carrier of embodiment 132, wherein said nanoparticle drug carrier contains a predefined ratio of IDO pathway inhibitor to ICD- inducer. [0156] Embodiment 134: The nanoparticle drug carrier according to any one of embodiments 132-133, wherein the IDO pathway inhibitor and the ICD inducer are synergistic in their activity against a cancer.
[0157] Embodiment 135 : The nanoparticle drug carrier according to any one of embodiments 132-134, wherein said drug carrier, when administered systemically, delivers an amount of an ICD inducer effective to induce or to facilitate induction of immunogenic cell death of a cancer cell.
[0158] Embodiment 136: The nanoparticle drug carrier according to any one of embodiments 132-135, wherein said drug carrier, when administered systemically, delivers an amount of IDO pathway inhibitor to partially or fully inhibit the IDO pathway at a cancer site. [0159] Embodiment 137: The nanoparticle drug carrier according to any one of embodiments 132-136, wherein said IDO pathway inhibitor, is disposed in said lipid bilayer and/or conjugated to a lipid comprising said lipid bilayer.
[0160] Embodiment 138: The nanoparticle drug carrier according to any one of embodiments 132-137, wherein said ICD inducer is disposed in said plurality of pores.
[0161] Embodiment 139: The nanoparticle drug carrier according to any one of embodiments 132-138, wherein said ICD inducer comprises a chemotherapeutic agent selected from the group consisting of oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
[0162] Embodiment 140: The nanoparticle drug carrier of embodiment 139, wherein said ICD inducer comprises oxaliplatin.
[0163] Embodiment 141 : The nanoparticle drug carrier according to any one of embodiments 132-140, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l -methyl - tryptophan (L-1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tiyptophan (L-1MT), methylthiohydantoin-dl -tryptophan (MTH-Trp, Necrostatin), β-carbolines (e.g., 3-butyl-P- carboline), Naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S- methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl- brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol-3-yl)ethyl]- S[(naphth-2-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-3-yl)methyl]- dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-4-yl)methyl]-dithiocarbamate, 5-bromo- brassinin, Phenylimidazole-based IDO inhibitors (e.g., 4-phenylimidazole), Exiguamine A, imidodicarbonimidic diamide,N-methyl-N'-9-phenanthrenyl-monohydrochloride
(NSC401366), INCB024360 (Epacadostat), l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDO 1 -derived peptide, NLG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole. [0164] Embodiment 142: The nanoparticle drug carrier of embodiment 141, wherein said IDO pathway inhibitor comprises 1 -methyl -tryptophan. [0165] Embodiment 143 : The nanoparticle drug carrier of embodiment 142, wherein said IDO pathway inhibitor comprises D-l-methyl-tiyptophan (indoximod).
[0166] Embodiment 144: The nanoparticle drug carrier of embodiment 142, wherein said IDO pathway inhibitor comprises L-l-methyl-tiyptophan (L-MT). [0167] Embodiment 145: The nanoparticle drug carrier of embodiment 142, wherein said IDO pathway inhibitor comprises a mixture of L-l-methyl-tiyptophan (L-MT) and D-l- methy 1 -tryptophan .
[0168] Embodiment 146: The nanoparticle drug carrier according to any one of embodiments 132-145, wherein said IDO pathway inhibitor is conjugated to a component of said lipid bilayer.
[0169] Embodiment 147: The nanoparticle drug carrier according to any one of embodiments 132-146, wherein said lipid bilayer comprises a phospholipid.
[0170] Embodiment 148: The nanoparticle drug carrier of embodiment 147, wherein said lipid bilayer comprises a phospholipid, cholesterol (CHOL), an mPEG phospholipid and/or an IDO pathway inhibitor lipid conjugate.
[0171] Embodiment 149: The nanoparticle drug carrier according to any one of embodiments 147-148, wherein said phospholipid comprises a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains. [0172] Embodiment 150: The nanoparticle drug carrier of embodiment 149, wherein said phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC),
distearoylphosphatidylcholine (DSPC), and diactylphosphatidylcholine (DAPC).
[0173] Embodiment 151 : The nanoparticle drug carrier of embodiment 149, wherein said phospholipid comprises a natural lipid selected from the group consisting of egg phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC).
[0174] Embodiment 152: The nanoparticle drug carrier of embodiment 149, wherein said phospholipid comprises an unsaturated fatty acid selected from the group consisting of l,2-dimyristoleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine,l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dieicosenoyl-sn- glycero-3 -phosphocholine. [0175] Embodiment 153 : The nanoparticle drug carrier according to any one of embodiments 147-152, wherein said lipid bilayer comprises an mPEG phospholipid with a phospholipid C14-C18 carbon chain, and a PEG molecular weight ranging from about 350 Da to 5000 Da. [0176] Embodiment 154: The nanoparticle drug carrier of embodiment 153, wherein said lipid bilayer comprises l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (DSPE- PEG).
[0177] Embodiment 155: The nanoparticle drug carrier of embodiment 154, wherein said lipid bilayer comprises DPSE-PEG2K.
[0178] Embodiment 156: The nanoparticle drug carrier according to any one of embodiments 147-155, wherein said IDO pathway inhibitor is conjugated to a moiety that forms a component of a lipid bilayer comprising a nanovesicle in aqueous solution and is provided in said lipid bilayer.
[0179] Embodiment 157: The nanoparticle drug carrier of embodiment 156, wherein said IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid.
[0180] Embodiment 158: The nanoparticle drug carrier according to any one of embodiments 156-157, wherein said IDO pathway inhibitor is conjugated directly to said moiety. [0181] Embodiment 159: The nanoparticle drug carrier according to any one of embodiments 156-157, wherein said IDO pathway inhibitor is conjugated to said moiety via a linker.
[0182] Embodiment 160: The nanoparticle drug carrier according to any one of embodiments 156-159, wherein said IDO pathway inhibitor is conjugated to PGHP. [0183] Embodiment 161 : The nanoparticle drug carrier according to any one of embodiments 156-159, wherein said IDO pathway inhibitor is conjugated to vitamin E.
[0184] Embodiment 162: The nanoparticle drug carrier according to any one of embodiments 156-159, wherein said IDO pathway inhibitor is conjugated to cholesterol (CHOL), or squalene. [0185] Embodiment 163 : The nanoparticle drug carrier according to any one of embodiments 156-159, wherein said IDO pathway inhibitor is conjugated to a fatty acid. [0186] Embodiment 164: The nanoparticle drug carrier of embodiment 163, wherein said i IDO pathway inhibitor is conjugated to oleic acid or docosahexaenoic acid.
[0187] Embodiment 165: The nanoparticle drug carrier of embodiment 163, wherein said IDO pathway inhibitor is conjugated to oleic acid or docosahexaenoic acid via an HO- (CH2)n=2-5-OH linker.
[0188] Embodiment 166: The nanoparticle drug carrier according to any one of embodiments 156-159, wherein said IDO pathway inhibitor is conjugated to a lipid.
[0189] Embodiment 167: The nanoparticle drug carrier of embodiment 166, wherein said IDO pathway inhibitor is conjugated to a phospholipid comprising said lipid bilayer.
[0190] Embodiment 168: The nanoparticle drug carrier of embodiment 167, wherein said IDO pathway inhibitor is conjugated to l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine (PL).
[0191] Embodiment 169: The nanoparticle drug carrier of embodiment 168, wherein the IDO athway inhibitor conjugated to a phospholipid has the structure:
Figure imgf000027_0001
[0192] Embodiment 170: The nanoparticle drug carrier of embodiment 169, wherein the 1-methyl-tiyptophan component of said conjugated IDO pathway inhibitor comprises a "D han (indoximod) characterized by the formula:
Figure imgf000027_0002
[0193] Embodiment 171 : The nanoparticle drug carrier of embodiment 169, wherein the 1-methyl-tiyptophan component of said conjugated IDO pathway inhibitor comprises an "L" isomer of 1 methyl-tyrptophan (L-1MT) characterized by the formula:
Figure imgf000028_0001
[0194] Embodiment 172: The nanoparticle drug carrier of embodiment 169, wherein the 1-methyl-tryptophan component of said conjugated IDO pathway inhibitor comprises a mixture of "D" and "L" isomers of 1-methyl-tryptophan.
[0195] Embodiment 173 : The nanoparticle drug carrier according to any one of embodiments 169-172, wherein said lipid bilayer comprises IND-PL/Chol/DSPE-PEG.
[0196] Embodiment 174: The nanoparticle drug carrier of embodiment 173, wherein said lipid bilayer comprises about 75% IND-PL, about 20% cholesterol, and about 5% DSPE- PEG2K.
[0197] Embodiment 175: The nanoparticle drug carrier according to any one of embodiments 132-174, wherein said lipid bilayer forms a substantially uniform and intact bilayer encompassing the entire nanoparticle.
[0198] Embodiment 176: The nanoparticle drug carrier drug carrier according to any one of embodiments 132-175, wherein said mesoporous silica nanoparticle is colloidally stable.
[0199] Embodiment 177: The nanoparticle drug carrier according to any one of embodiments 132-176, wherein said mesoporous silica has: an average pore size that ranges from about 1 to about 20 nm, or from about 1 to about 10 nm, or from about 2 to about 8 nm; and an average size ranging from about 50 nm up to about 300 nm, or from about 50 up to about 200 nm, or from about 50 up to about 150 nm, or from about 50 up to about 100 nm, or from about 50 up to about 80 nm, or from about 50 up to about 70 nm, or from about 60 up to about 70 nm.
[0200] Embodiment 178: The nanoparticle drug carrier according to any one of embodiments 132-177, wherein when the drug in the nanocarrier is a weak base, said carrier comprises a cargo-trapping agent. [0201] Embodiment 179: The nanoparticle drug carrier of embodiment 178, wherein said cargo trapping agent before reaction with the drug loaded in the nanoparticle drug carrier, is selected from the group consisting of triethylammonium sucrose octasulfate (TEA8SOS), ( H4)2S04, an ammonium salt, a trimethylammonium salt, and a
triethylammonium salt.
[0202] Embodiment 180: The nanoparticle drug carrier of embodiment 179, wherein cargo-trapping agent before reaction with said drug is triethylammonium sucrose octasulfate (TEA8SOS).
[0203] Embodiment 181 : The nanoparticle drug carrier according to any one of embodiments 179-180, wherein said drug is protonated and trapped in said pores as a gel-like precipitate in association of SOS8".
[0204] Embodiment 182: The nanoparticle drug carrier according to any one of embodiments 132-181, wherein said drug carrier is conjugated to a moiety selected from the group consisting of a targeting moiety, a fusogenic peptide, and a transport peptide. [0205] Embodiment 183 : The nanoparticle drug carrier of embodiment 182, wherein said drug carrier is conjugated to a peptide that binds a receptor on a cancer cell or tumor blood vessel.
[0206] Embodiment 184: The nanoparticle drug carrier of embodiment 183, wherein said drug carrier is conjugated to an iRGD peptide. [0207] Embodiment 185: The nanoparticle drug carrier of embodiment 183, wherein said drug carrier is conjugated to a targeting peptide shown in Table 5.
[0208] Embodiment 186: The nanoparticle drug carrier according to any one of embodiments 182-185, wherein said drug carrier is conjugated to transferrin, and/or ApoE, and/or folate. [0209] Embodiment 187: The nanoparticle drug carrier according to any one of embodiments 182-186, wherein said drug carrier is conjugated to a targeting moiety that comprises an antibody that binds to a cancer marker.
[0210] Embodiment 188: The nanoparticle drug carrier of embodiment 187, wherein said antibody comprises a full-length antibody (intact immunoglobuloin). [0211] Embodiment 189: The nanoparticle drug carrier of embodiment 187, wherein said antibody comprises an antibody fragment. [0212] Embodiment 190: The nanoparticle drug carrier of embodiment 187, wherein said antibody is selected from the group consisting of an intact immunoglobulin, an F(ab)'2, a Fab, a single chain antibody, a diabody, an affibody, a unibody, and a nanobody.
[0213] Embodiment 191 : The nanoparticle drug carrier according to any one of embodiments 187-190, wherein said antibody comprise an antibody that binds a cancer marker shown in Table 4.
[0214] Embodiment 192: The nanoparticle drug carrier according to any one of embodiments 132-191, wherein said drug carriers in suspension are stable for at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months when stored at 4°C.
[0215] Embodiment 193 : The nanoparticle drug carrier according to any one of embodiments 132-192, wherein said nanoparticle drug carrier forms a stable suspension on rehydration after lyophilization.
[0216] Embodiment 194: The nanoparticle drug carrier according to any one of embodiments 132-193, wherein said nanoparticle drug carriers, show reduced drug toxicity as compared to free drug and/or drug in a liposome.
[0217] Embodiment 195: The nanoparticle drug carrier according to any one of embodiments 132-194, wherein said nanoparticle drug carrier has colloidal stability in physiological fluids with pH 7.4 and remains monodisperse to allow systemic biodistribution and is capable of entering a disease site by vascular leakage (EPR effect) or transcytosis.
[0218] Embodiment 196: A nanomaterial carrier for the combined delivery of an inhibitor of the IDO pathway and an inducer of immunogenic cell death (ICD), said nanomaterial carrier comprising:
[0219] a nanomaterial that induces ICD; and
[0220] a lipid or lipid formulation comprising an IDO pathway inhibitor where said lipid or lipid formulation is disposed on the surface of said nanomaterial.
[0221] Embodiment 197: The nanomaterial carrier of embodiment 196, wherein said lipid or lipid formulation fully encapsulates said nanomaterial.
[0222] Embodiment 198: The nanomaterial carrier according to any one of embodiments 196-197, wherein said lipid or lipid formulation is not a lipid bilayer. [0223] Embodiment 199: The nanomaterial carrier according to any one of embodiments 196-197, wherein said lipid or lipid formulation comprises a lipid bilayer.
[0224] Embodiment 200: The nanomaterial carrier according to any one of embodiments 196-199, wherein said nanomaterial comprises a material selected from the group consisting of selected from the group consisting of CuO, Cu20, Sb203, As203, Bi203, P203, ZnO, Ti02, graphene oxide, and 2D materials other than graphene or graphene oxide.
[0225] Embodiment 201 : The nanomaterial carrier of embodiment 200, wherein said nanomaterial comprises copper oxide (CuO).
[0226] Embodiment 202: The nanomaterial carrier according to any one of embodiments 200-201, wherein said nanomaterial comprises Sb203.
[0227] Embodiment 203 : The nanomaterial carrier according to any one of embodiments 200-202, wherein said material comprise a 2D nanomaterial.
[0228] Embodiment 204: The nanomaterial carrier of embodiment 203, said 2D nanomaterial comprises a material selected from the group consisting of graphene, graphyne, borophene, germanene, silicene, Si2BN, stanene, phosphorene, bismuthene, molybdenite, metals, and a 2D supracrystal.
[0229] Embodiment 205 : The nanomaterial carrier according to any one of embodiments 200-204, wherein said nanomaterial comprises graphene oxide (GO).
[0230] Embodiment 206: The nanomaterial carrier according to any one of embodiments 196-205, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l -methyl - tryptophan (L-1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tiyptophan (L-1MT), methylthiohydantoin-dl -tryptophan (MTH-Trp, Necrostatin), β-carbolines (e.g., 3-butyl-P- carboline), Naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S- methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl- brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol-3-yl)ethyl]- S[(naphth-2-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-3-yl)methyl]- dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-4-yl)methyl]-dithiocarbamate, 5-bromo- brassinin, Phenylimidazole-based IDO inhibitors (e.g., 4-phenylimidazole), Exiguamine A, imidodicarbonimidic diamide,N-methyl-N'-9-phenanthrenyl-monohydrochloride
(NSC401366), INCB024360 (Epacadostat), l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDOl -derived peptide, LG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole.
[0231] Embodiment 207: The nanomaterial carrier according to any one of embodiments 196-205, wherein said IDO pathway inhibitor comprises 1 -methyl tryptophan.
[0232] Embodiment 208: The nanomaterial carrier of embodiment 207, wherein said
IDO pathway inhibitor comprises a D isomer of 1 -methyl tryptophan.
[0233] Embodiment 209: The nanomaterial carrier of embodiment 207, wherein said
IDO pathway inhibitor comprises an L isomer of 1 -methyl tryptophan. [0234] Embodiment 210: The nanomaterial carrier of embodiment 207, wherein said
IDO pathway inhibitor comprises a mixture of D and L isomers of 1 -methyl tryptophan.
[0235] Embodiment 211 : The nanomaterial carrier to any one of embodiments 196-
210, wherein said IDO pathway inhibitor is conjugated to said lipid or to a component of said lipid formulation. [0236] Embodiment 212: The nanomaterial carrier of embodiment 211, wherein said
IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid.
[0237] Embodiment 213 : The nanomaterial carrier according to any one of embodiments 211-212, wherein said IDO pathway inhibitor is conjugated directly to said moiety.
[0238] Embodiment 214: The nanomaterial carrier according to any one of embodiments 211-212, wherein said IDO pathway inhibitor is conjugated to said moiety via a linker.
[0239] Embodiment 215: The nanomaterial carrier according to any one of embodiments 212-214, wherein said IDO pathway inhibitor is conjugated to PGHP.
[0240] Embodiment 216: The nanomaterial carrier according to any one of embodiments 212-214, wherein said IDO pathway inhibitor is conjugated to vitamin E.
[0241] Embodiment 217: The nanomaterial carrier according to any one of embodiments 212-214, wherein said IDO pathway inhibitor is conjugated to cholesterol (CHOL). [0242] Embodiment 218: The nanomaterial carrier according to any one of embodiments 212-214, wherein said IDO pathway inhibitor is conjugated to a fatty acid.
[0243] Embodiment 219: The nanomaterial carrier of embodiment 218, wherein said inhibitor of the IDO pathway is conjugated to oleic acid or docosahexaenoic acid. [0244] Embodiment 220: The nanomaterial carrier of embodiment 219, wherein said
IDO pathway inhibitor is conjugated to oleic acid or docosahexaenoic acid via an HO- (CH2)n=2-5-OH linker.
[0245] Embodiment 221 : The nanomaterial carrier according to any one of embodiments 212-214, wherein said IDO pathway inhibitor is conjugated to a lipid. [0246] Embodiment 222: The nanomaterial carrier of embodiment 221, wherein said
IDO pathway inhibitor is conjugated to a phospholipid.
[0247] Embodiment 223 : The nanomaterial carrier of embodiment 222, wherein said inhibitor of the IDO pathway is conjugated to a phospholipid comprising a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains.
[0248] Embodiment 224: The nanomaterial carrier of embodiment 223, wherein said phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC),
distearoylphosphatidylcholine (DSPC), and diactylphosphatidylcholine (DAPC). [0249] Embodiment 225: The nanomaterial carrier of embodiment 223, wherein said phospholipid comprises a natural lipid selected from the group consisting of egg
phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC).
[0250] Embodiment 226: The nanomaterial carrier of embodiment 223, wherein said phospholipid comprises an unsaturated fatty acid selected from the group consisting of 1,2- dimyristoleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine,l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dieicosenoyl-sn- glycero-3 -phosphocholine.
[0251] Embodiment 227: The nanomaterial carrier of embodiment 223, wherein said phospholipid comprises l-palmitoyl-2-hydroxy-sn-glycero-3 -phosphocholine. [0252] Embodiment 228: The nanomaterial carrier according to any one of embodiments 196-227, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of of D-l-methyl-tryptophan (indoximod, D-1MT), L-l -methyl - tryptophan (L-1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tryptophan (L-1MT), methylthiohydantoin-dl -tryptophan (MTH-Trp, Necrostatin), β-carbolines (e.g., 3-butyl-P- carboline), Naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S- methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl- brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol-3-yl)ethyl]- S[(naphth-2-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-3-yl)methyl]- dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-4-yl)methyl]-dithiocarbamate, 5-bromo- brassinin, Phenylimidazole-based IDO inhibitors (e.g., 4-phenylimidazole), Exiguamine A, imidodicarbonimidic diamide,N-methyl-N'-9-phenanthrenyl-monohydrochloride
(NSC401366), INCB024360 (Epacadostat), l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDO l -derived peptide, NLG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole.
[0253] Embodiment 229: The nanomaterial carrier according to any one of embodiments 196-227, wherein said IDO pathway inhibitor comprises 1 -methyl -tryptophan.
[0254] Embodiment 230: The nanomaterial carrier of embodiment 229, wherein said
1 -methyl -tryptophan comprises a D isomer of 1-methyl-tiyptophan. [0255] Embodiment 231 : The nanomaterial carrier of embodiment 229, wherein said
1 -methyl -tryptophan comprises an L isomer of 1 -methyl -tryptophan.
[0256] Embodiment 232: The nanomaterial carrier of embodiment 229, wherein said
1 -methyl -tryptophan comprises a mixture of D and L isomers of 1 -methyl -tryptophan.
[0257] Embodiment 233 : The nanomaterial carrier of embodiment 229, wherein said 1 -methyl -tryptophan is conjugated to l -palmitoy l -2-hydroxy-s«-glycero-3-phosphocholine.
[0258] Embodiment 234: The nanomaterial carrier of embodiment 229, wherein said
1 -methyl -tryptophan conjugated to l-palmitoy l -2-hydroxy-s«-glycero-3-phosphocholine com rises a compound having the structure:
Figure imgf000035_0001
[0259] Embodiment 235: The nanomaterial carrier of embodiment 234, wherein the
1 -methyl -tryptophan component of said conjugated IDO pathway inhibitor comprises a "D" isom r of 1 methyl-tyrptophan (indoximod) characterized by the formula:
Figure imgf000035_0002
[0260] Embodiment 236: The nanovesicle drug carrier of embodiment 234, wherein the 1-methyl-tiyptophan component of said conjugated IDO pathway inhibitor comprises an "L" isomer of 1 methyl-tyrptophan (L-1MT) characterized by the formula:
Figure imgf000035_0003
[0261] Embodiment 237: The nanovesicle drug carrier of embodiment 234, wherein the 1-methyl-tiyptophan component of said conjugated IDO pathway inhibitor comprises a mixture of "D" and "L" isomers of 1 -methyl -tryptophan.
[0262] Embodiment 238: The nanomaterial carrier according to any one of embodiments 196-237, wherein said ICD inducer and said IDO pathway inhibitor are synergistic in their activity on a cancer. [0263] Embodiment 239: The nanomaterial carrier according to any one of embodiments 196-238, wherein said effective amount of said ICD-inducer is an amount effective to elevate calreticulin (CRT) expression in cancer cells at the tumor site.
[0264] Embodiment 240: The nanomaterial carrier according to any one of embodiments 196-239, wherein said effective amount of said ICD-inducer is an amount effective to elevate expression and/or release of HMGB1 and/or induction of ATP release in cancer cells at the tumor site.
[0265] Embodiment 241 : The nanomaterial carrier according to any one of embodiments 196-240, wherein said drug carrier is conjugated to a moiety selected from the group consisting of a targeting moiety, a fusogenic peptide, and a transport peptide.
[0266] Embodiment 242: The nanomaterial carrier of embodiment 241, wherein said drug carrier is conjugated to a peptide that binds a receptor on a cancer cell or tumor blood vessel at the tumor site.
[0267] Embodiment 243 : The nanomaterial carrier of embodiment 242, wherein said drug carrier is conjugated to an iRGD peptide.
[0268] Embodiment 244: The nanomaterial carrier of embodiment 242, wherein said drug carrier is conjugated to a targeting peptide shown in Table 5.
[0269] Embodiment 245: The nanomaterial carrier according to any one of embodiments 241-244, wherein said drug carrier is conjugated to transferrin, and/or ApoE, and/or folate.
[0270] Embodiment 246: The nanomaterial carrier according to any one of embodiments 241-245, wherein said drug carrier is conjugated to a targeting moiety that comprises an antibody that binds to a cancer marker.
[0271] Embodiment 247: The nanomaterial carrier of embodiment 246, wherein said drug carrier is conjugated to a targeting moiety that comprises an antibody that binds a cancer marker shown in Table 4.
[0272] Embodiment 248: A pharmaceutical formulation said formulation comprising:
[0273] a plurality of nanovesicle drug carriers according to any one of embodiments 1-59, and/or a plurality of nanoparticle drug carriers according to any one of embodiments 132-195, and/or a plurality of nanomaterial carriers according to any one of embodiments 196-254; and
[0274] a pharmaceutically acceptable carrier.
[0275] Embodiment 249: The formulation of embodiment 248, wherein said formulation comprises a plurality of nanoparticle drug carriers according to any one of embodiments 132-195.
[0276] Embodiment 250: The formulation of embodiment 248, wherein said formulation comprises a plurality of nanovesicle drug carriers according to any one of embodiments 1-59.
[0277] Embodiment 251 : The formulation of embodiment 248, wherein said formulation comprises a plurality of nanomaterial carriers according to any one of embodiments 196-254.
[0278] Embodiment 252: The formulation according to any one of embodiments of embodiments 248-251, wherein said formulation is an emulsion, dispersion, or suspension.
[0279] Embodiment 253 : The formulation of embodiment 252, wherein said suspension, emulsion, or dispersion is stable for at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months when stored at 4°C.
[0280] Embodiment 254: The formulation according to any one of embodiments
248-253, wherein: the nanoscale drug carriers in said formulation show a substantially unimodal size distribution; and/or the drug carriers in said suspension, emulsion, or dispersion shows a PDI less than about 0.2, or less than about 0.1.
[0281] Embodiment 255 : The formulation according to any one of embodiments
248-254, wherein said formulation is formulated for administration via a route selected from the group consisting of intravenous administration, intraarterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation (including intranasal and intratracheal delivery, intracranial administration via a cannula, and subcutaneous or intramuscular depot deposition.
[0282] Embodiment 256: The formulation according to any one of embodiments
248-254, wherein said formulation is a sterile injectable. [0283] Embodiment 257: The formulation according to any one of embodiments
248-256, wherein said formulation is a unit dosage formulation. [0284] Embodiment 258: A method of treating a cancer in a mammal, said method comprising:
[0285] administering to an intra-tumoral or peri-tumoral site an effective amount of an IDO pathway inhibitor in conjunction with an effective amount of an agent that induces immunogenic cell death (ICD) (an ICD-inducer).
[0286] Embodiment 259: The method of embodiment 258, wherein said ICD inducer and said IDO pathway inhibitor are synergistic in their activity on said cancer.
[0287] Embodiment 260: The method according to any one of embodiments 258-
259, wherein said effective amount of said ICD-inducer is an amount effective to elevate calreticulin (CRT) expression in cells of said cancer at the tumor site.
[0288] Embodiment 261 : The method according to any one of embodiments 258-
260, wherein said effective amount of said ICD-inducer is an amount effective to elevate expression and/or release of HMGB l and/or induction of ATP release in cancer cells at the tumor site. [0289] Embodiment 262: The method according to any one of embodiments 258-
261, wherein said IDO pathway inhibitor and said ICD inducer are provided as a nanoparticle drug carrier according to any one of embodiments 132-195.
[0290] Embodiment 263 : The method according to any one of embodiments 258-
261, wherein said IDO pathway inhibitor and said ICD inducer are provided as a
nanomaterial carrier according to any one of embodiments 196-254.
[0291] Embodiment 264: The method according to any one of embodiments 258-
261, wherein said IDO pathway inhibitor is conjugated to a moiety that forms a component of a lipid bilayer comprising a vesicle in aqueous solution.
[0292] Embodiment 265: The method of embodiment 264, wherein said IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid.
[0293] Embodiment 266: The method according to any one of embodiments 264-
265, wherein said IDO pathway inhibitor is conjugated directly to said moiety.
[0294] Embodiment 267: The method according to any one of embodiments 264- 265, wherein said IDO pathway inhibitor is conjugated to said moiety via a linker. [0295] Embodiment 268: The method according to any one of embodiments 265-
267, wherein said IDO pathway inhibitor is conjugated to PGHP.
[0296] Embodiment 269: The method according to any one of embodiments 265-
267, wherein said IDO pathway inhibitor is conjugated to vitamin E. [0297] Embodiment 270: The method according to any one of embodiments 265-
267, wherein said IDO pathway inhibitor is conjugated to cholesterol (CHOL).
[0298] Embodiment 271 : The method according to any one of embodiments 265-
267, wherein said IDO pathway inhibitor is conjugated to a fatty acid.
[0299] Embodiment 272: The method of embodiment 271, wherein said IDO pathway inhibitor is conjugated to oleic acid or docosahexaenoic acid.
[0300] Embodiment 273 : The method of embodiment 272, wherein said IDO pathway inhibitor is conjugated to oleic acid or docosahexaenoic acid via an HO-(CH2)n=2.5- OH linker.
[0301] Embodiment 274: The method according to any one of embodiments 265- 267, wherein said IDO pathway inhibitor is conjugated to a lipid.
[0302] Embodiment 275: The method of embodiment 274, wherein said IDO pathway inhibitor is conjugated to a phospholipid.
[0303] Embodiment 276: The method of embodiment 275, wherein said IDO pathway inhibitor is conjugated to a phospholipid comprising a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains.
[0304] Embodiment 277: The method of embodiment 276, wherein said
phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC),
distearoylphosphatidylcholine (DSPC), and diactylphosphatidylcholine (DAPC).
[0305] Embodiment 278: The method of embodiment 276, wherein said
phospholipid comprises a natural lipid selected from the group consisting of egg
phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC).
[0306] Embodiment 279: The method of embodiment 276, wherein said
phospholipid comprises an unsaturated fatty acid selected from the group consisting of 1,2- dimyristoleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine, l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dieicosenoyl-sn- glycero-3 -phosphocholine.
[0307] Embodiment 280: The method of embodiment 276, wherein said
phospholipid comprises l-palmitoy l -2-hydroxy-sn-glycero-3 -phosphocholine.
[0308] Embodiment 281 : The method according to any one of embodiments 258-
280, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of of D-l-methyl-tryptophan (indoximod, D-1MT), L-l-methyl-tryptophan (L- 1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tryptophan (L-1MT),
methylthiohydantoin-dl-tryptophan (MTH-Trp, Necrostatin), β-carbolines (e.g., 3-butyl-P- carboline), Naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S- methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl- brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol-3-yl)ethyl]- S[(naphth-2-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-3-yl)methyl]- dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-4-yl)methyl]-dithiocarbamate, 5-bromo- brassinin, Phenylimidazole-based IDO inhibitors (e.g., 4-phenylimidazole), Exiguamine A, imidodicarbonimidic diamide,N-methyl-N'-9-phenanthrenyl-monohydrochloride
(NSC401366), INCB024360 (Epacadostat), l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDO l -derived peptide, NLG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole.
[0309] Embodiment 282: The method according to any one of embodiments 258-
280, wherein said IDO pathway inhibitor comprises 1-methyl-tiyptophan. [0310] Embodiment 283 : The method of embodiment 282, wherein said IDO pathway inhibitor comprises a substantially pure D enantiomer of 1-methyl-tiyptophan.
[0311] Embodiment 284: The method of embodiment 282, wherein said inhibitor of
IDO pathway inhibitor comprises a substantially pure L enantiomer of 1-methyl-tiyptophan.
[0312] Embodiment 285 : The method of embodiment 284, wherein said 1-methyl- tryptophan is conjugated to l -palmitoy l-2-hydroxy-5«-glycero-3 -phosphocholine.
[0313] Embodiment 286: The method of embodiment 285, wherein said 1-methyl- tryptophan conjugated to l -palmitoy l -2-hydroxy-5«-glycero-3 -phosphocholine comprises a com ound having the structure:
Figure imgf000041_0001
[0314] Embodiment 287: The method of embodiment 286, wherein the 1-methyl- tryptophan component of said conjugated IDO pathway inhibitor comprises a "D" isomer methyl-tyrptophan (indoximod) characterized by the formula:
Figure imgf000041_0002
[0315] Embodiment 288: The method of embodiment 286, wherein the 1-methyl- tryptophan component of said conjugated IDO pathway inhibitor comprises an "L" isomer of 1 meth -tyrptophan (L-1MT) characterized by the formula:
Figure imgf000041_0003
[0316] Embodiment 289: The method of embodiment 286, wherein the 1-methyl- tryptophan component of said conjugated IDO pathway inhibitor comprises a mixture of "D" and "L" isomers of 1-methyl-tiyptophan.
[0317] Embodiment 290: The method according to any one of embodiments 274-
289, wherein the lipid-conjugated IDO pathway inhibitor forms a component of a vesicle.
[0318] Embodiment 291 : The method according to any one of embodiments 258-
290, wherein said agent that induces immunogenic cell death (ICD) comprises an agent selected from the group consisting of oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
[0319] Embodiment 292: The method of embodiment 291, wherein said agent that induces immunogenic cell death (ICD) comprises oxaliplatin. [0320] Embodiment 293 : The method according to any one of embodiments 258-
290, wherein said agent that induces immunogenic cell death (ICD) comprises or contains a nanomaterial that induces ICD.
[0321] Embodiment 294: The method of embodiment 293, wherein said
nanomaterial forms a nanoparticle. [0322] Embodiment 295: The method of embodiment 293, wherein said
nanomaterial comprises a core-shell nanoparticle.
[0323] Embodiment 296: The method of embodiment 293, wherein said
nanomaterial comprises a doped nanoparticle.
[0324] Embodiment 297: The method according to any one of embodiments 292- 296, wherein said agent that induces immunogenic cell death (ICD) comprises a nanomaterial selected from the group consisting of CuO, Cu20, Sb203, As203, Bi203, P203, ZnO, Ti02, graphene oxide, and 2D materials other than graphene or graphene oxide.
[0325] Embodiment 298: The method of embodiment 297, wherein said
nanomaterial comprises copper oxide (CuO). [0326] Embodiment 299: The method of embodiment 297, wherein said
nanomaterial comprises Sb203.
[0327] Embodiment 300: The method of embodiment 297, wherein said material comprise a 2D nanomaterial.
[0328] Embodiment 301 : The method of embodiment 300, said 2D nanomaterial comprise a material selected from the group consisting of graphene, graphyne, borophene, germanene, silicene, si2bn, stanene, phosphorene, bismuthene, molybdenite, metals, and a 2D supracrystal.
[0329] Embodiment 302: The method of embodiment 301, wherein said
nanomaterial comprises graphene oxide (GO). [0330] Embodiment 303 : The method according to any one of embodiments 258-
302, wherein agent that induces immunogenic cell death (ICD) comprises an oncolytic virus.
[0331] Embodiment 304: The method of embodiment 303, wherein said oncolytic virus comprises a virus selected from the group consisting of Parvovirus (e.g., H-PV), Adenovirus (e.g. , hTERT-Ad, Ad5/3-D24-GMCSF), Herpes simplex virus (e.g., G207, HSV- 1716, T-VEC, HSV-2 ΔΡΚ mutant), Poxvirus (e.g., vSP, vvDDPexa-Vec), Arbovirus (e.g., VSV-GFP Indiana serotype, VSVgm-icv), and Paramyxovirus (e.g., MV-eGFP (Edmonston strain)).
[0332] Embodiment 305 : The method according to any one of embodiments 258- 304, wherein said IDO pathway inhibitor is administered before said agent that induces immunogenic cell death (ICD) (ICD-inducer).
[0333] Embodiment 306: The method according to any one of embodiments 258-
304, wherein said IDO pathway inhibitor is administered after said agent that induces immunogenic cell death (ICD) (ICD-inducer). [0334] Embodiment 307: The method according to any one of embodiments 258-
304, wherein said IDO pathway inhibitor inhibitor is administered simultaneously with said agent that induces immunogenic cell death (ICD) (ICD-inducer).
[0335] Embodiment 308: The method of embodiment 307, wherein said IDO pathway inhibitor and said agent that induces immunogenic cell death (ICD) (ICD-inducer) are administered as a combined formulation.
[0336] Embodiment 309: The method according to any one of embodiments 258-
308, wherein said IDO pathway inhibitor and/or said agent that induces immunogenic cell death (ICD) (ICD-inducer) is delivered into a tumor.
[0337] Embodiment 310: The method according to any one of embodiments 258- 309, wherein said IDO pathway inhibitor and/or said agent that induces immunogenic cell death (ICD) (ICD-inducer) is delivered into a peri-tumoral site.
[0338] Embodiment 31 1 : The method according to any one of embodiments 258-
310, wherein said IDO pathway inhibitor and/or said agent that induces immunogenic cell death (ICD) (ICD-inducer) is delivered into a post-surgical site. [0339] Embodiment 312: The method according to any one of embodiments 258-
31 1, wherein said IDO pathway inhibitor and/or said agent that induces immunogenic cell death (ICD) (ICD-inducer) is administered via a method selected from the group consisting of injection, delivery via a cannula, depot deposition, and washing of a surgical site.
[0340] Embodiment 313 : The method according to any one of embodiments 258-
312, wherein said cancer is selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Adrenocortical carcinoma, Kaposi sarcoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma,
ependymoma, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, kaposi sarcoma, kidney cancer (e.g., renal cell, Wilm's tumor, and other kidney tumors), langerhans cell histiocytosis, laryngeal cancer, leukemia, acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myelogenous (CML), hairy cell, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., childhood, non-small cell, small cell), lymphoma (e.g., AIDS-related, Burkitt (e.g., non-Hodgkin lymphoma), cutaneous T-Cell (e.g., mycosis fungoides, Sezary syndrome), Hodgkin, non- Hodgkin, primary central nervous system (CNS)), macroglobulinemia, Waldenstrom, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma (e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, Myelogenous Leukemia, Chronic (CML), multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Ewing, Kaposi, osteosarcoma, rhadomyosarcoma, soft tissue, uterine), Sezary syndrome, skin cancer (e.g., melanoma, merkel cell carcinoma, basal cell carcinoma, nonmelanoma), small intestine cancer, squamous cell carcinoma, squamous neck cancer with occult primary, stomach (gastric) cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom
macroglobulinemia, and Wilm's tumor.
[0341] Embodiment 314: The method according to any one of embodiments 258- 312, wherein said cancer is pancreatic ductal adenocarcinoma (PDAC).
[0342] Embodiment 315 : The method according to any one of embodiments 258-
314, wherein said mammal is a human.
[0343] Embodiment 316: The method according to any one of embodiments 258-
314, wherein said mammal is a non-human mammal. [0344] Embodiment 317: A method of treating a cancer, said method comprising: administering to a subject in need thereof an effective amount of a nanoparticle drug carrier according to any one of embodiments 132-195; and/or a nanomaterial carrier according to any one of embodiments 196-254.
[0345] Embodiment 318: The method of embodiment 317, wherein said method comprises administering an effective amount of a nanoparticle drug carrier according to any one of embodiments 132-195. [0346] Embodiment 319: The method of embodiment 317, wherein said method comprises administering an effective amount of ICD-inducing nanomaterial carriers according to any one of embodiments 196-254.
[0347] Embodiment 320: The method according to any one of embodiments 317- 319, wherein the ICD inducer and the IDO pathway inhibitor are synergistic in their activity against said cancer.
[0348] Embodiment 321 : The method according to any one of embodiments 317-
320, wherein said nanoparticle drug carrier and/or said pharmaceutical formulation is a primary therapy in a chemotherapeutic regimen. [0349] Embodiment 322: The method according to any one of embodiments 317-
320, wherein said nanoparticle drug carrier and/or said pharmaceutical formulation is an adjunct therapy in a treatment regime that additionally comprises chemotherapy using another chemotherapeutic agent, and/or surgical resection of a tumor mass, and/or radiotherapy. [0350] Embodiment 323 : The method according to any one of embodiments 317-
320, wherein said nanoparticle drug carrier and/or said pharmaceutical formulation is a component in a multi-drug chemotherapeutic regimen.
[0351] Embodiment 324: The method according to any one of embodiments 317-
323, wherein said cancer is pancreatic ductal adenocarcinoma (PDAC). [0352] Embodiment 325 : The method according to any one of embodiments 317-
323, wherein said cancer is a cancer selected from the group consisting of acute
lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi sarcoma, lymphoma), anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer {e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors {e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kaposi sarcoma, kidney cancer {e.g., renal cell, Wilm's tumor, and other kidney tumors), langerhans cell histiocytosis, laryngeal cancer, leukemia, acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myelogenous (CML), hairy cell, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer {e.g., childhood, non-small cell, small cell), lymphoma {e.g., AIDS-related, Burkitt {e.g., non-Hodgkin lymphoma), cutaneous T-Cell {e.g., mycosis fungoides, Sezary syndrome), Hodgkin, non-Hodgkin, primary central nervous system (CNS)), macroglobulinemia, Waldenstrom, male breast cancer, malignant fibrous
histiocytoma of bone and osteosarcoma, melanoma {e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple
myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes,
Myelogenous Leukemia, Chronic (CML), multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer , pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma {e.g., Ewing,
Kaposi, osteosarcoma, rhadomyosarcoma, soft tissue, uterine), Sezary syndrome, skin cancer {e.g., melanoma, merkel cell carcinoma, basal cell carcinoma, nonmelanoma), small intestine cancer, squamous cell carcinoma, squamous neck cancer with occult primary, stomach (gastric) cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom
macroglobulinemia, and Wilm's tumor.
[0353] Embodiment 326: The method according to any one of embodiments 317- 325, wherein said nanoparticle drug carrier is not conjugated to an iRGD peptide and the nanoparticle drug carrier is administered in conjunction with an iRGD peptide.
[0354] Embodiment 327: The method according to any one of embodiments 317-
326, wherein said nanoparticle drug carrier is administered in conjunction with administration of an immune checkpoint inhibitor. [0355] Embodiment 328: The method of embodiment 327, wherein said immune checkpoint inhibitor comprises an inhibitor of PD-1, PD-Ll, PD-L2, PD-L3, PD-L4, CTLA- 4, LAG3, B7-H3, B7-H4, KIR and/or TIM3.
[0356] Embodiment 329: The method of embodiment 328, wherein said checkpoint inhibitor comrpies an antibody that inhibits a moiety selected from the group consisting of PD-1, PD-Ll, and CTLA4.
[0357] Embodiment 330: The method of embodiment 329, wherein said antibody comprises an antibody that inhibits PD-1.
[0358] Embodiment 331 : The method of embodiment 330, wherein said antibody comprises Pembrolizumab (Keytruda), or Nivolumab (Opdivo). [0359] Embodiment 332: The method of embodiment 329, wherein said antibody comprises an antibody that inhibits PD-Ll .
[0360] Embodiment 333 : The method of embodiment 332, wherein said antibody comprises Atezolizumab (Tecentriq), Avelumab (Bavencio), or Durvalumab (Imfinzi).
[0361] Embodiment 334: The method of embodiment 329, wherein said antibody comprises an antibody that inhibits CTLA-4.
[0362] Embodiment 335: The method of embodiment 334, wherein said antibody comprises Ipilimumab (Yervoy).
[0363] Embodiment 336: The method according to any one of embodiments 327-
335, wherein the activity of said nanoparticle drug carrier and said immune checkpoint inhibitor is synergistic. [0364] Embodiment 337: A method for the treatment and/or prevention of a cancer in a mammal, said method comprising: providing cancer cells in which immunogenic cell death (ICD) has been induced ex vivo; and vaccinating said mammal with said cells, where said vaccination induces an anti-cancer immunogenic response. [0365] Embodiment 338: The method of embodiment 337, wherein said
immunogenic cell death is induced by contacting said cancer cells with a chemotherapeutic agent that induces immunogenic cell death.
[0366] Embodiment 339: The method of embodiment 338, wherein said
immunogenic cell death (ICD) is induced by contacting said cells with a chemotherapeutic agent selected from the group consisting of oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
[0367] Embodiment 340: The method according to any one of embodiments 337-
339, wherein said immunogenic cell death (ICD) is induced by contacting said cells with oxaliplatin.
[0368] Embodiment 341 : The method according to any one of embodiments 337-
340, wherein said immunogenic cell death (ICD) is induced by contacting said cells with doxorubicin.
[0369] Embodiment 342: The method according to any one of embodiments 337- 340, wherein said immunogenic cell death (ICD) is induced by contacting said cells with a bioreactive nanomaterial that induces ICD.
[0370] Embodiment 343 : The method of embodiment 342, wherein said
immunogenic cell death (ICD) is induced by contacting said cells with a composition that contains or comprises a bioreactive nanomaterial that induces ICD. [0371] Embodiment 344: The method according to any one of embodiments 342-
343, wherein said ICD-inducing nanomaterial comprises a material selected from the group consisting of CuO, Cu20, Sb203, As203, Bi203, P203, ZnO, Ti02, graphene oxide, and 2D materials other than graphene or graphene oxide.
[0372] Embodiment 345: The method of embodiment 344, wherein said
nanomaterial comprises copper oxide (e.g. CuO). [0373] Embodiment 346: The method according to any one of embodiments 344-
345, wherein said nanomaterial comprises Sb2C>3.
[0374] Embodiment 347: The method of embodiment 344, wherein said material comprise a 2D nanomaterial. [0375] Embodiment 348: The method of embodiment 347, said 2D nanomaterial comprise a material selected from the group consisting of graphene, graphyne, borophene, germanene, silicene, si2bn, stanene, phosphorene, bismuthene, molybdenite, metals, and a 2D supracrystal.
[0376] Embodiment 349: The method of embodiment 348, wherein said
nanomaterial comprises graphene oxide (GO).
[0377] Embodiment 350: The method according to any one of embodiments 342-
349, wherein said nanomaterial comprises a nanoparticle.
[0378] Embodiment 351 : The method of embodiment 348, wherein said
nanomaterial comprises a core-shell nanoparticle. [0379] Embodiment 352: The method of embodiment 348, wherein said
nanomaterial comprises a doped nanoparticle.
[0380] Embodiment 353 : The method according to any one of embodiments 337-
352, wherein said immunogenic cell death is induced by exposure to ICD-inducing radiation (e.g., gamma radiation, UVC, etc.), or ICD-inducing photodynamic therapy. [0381] Embodiment 354: The method according to any one of embodiments 337-
353, wherein said immunogenic cell death is induced by infecting said cells with an oncolytic virus.
[0382] Embodiment 355: The method of embodiment 354, wherein said oncolytic virus is selected from the group consisting of Parvovirus (e.g., H-PV), Adenovirus (e.g., hTERT-Ad, Ad5/3-D24-GMCSF), Herpes simplex virus (e.g., G207, HSV-1716, T-VEC, HSV-2 ΔΡΚ mutant), Poxvirus (e.g., vSP, vvDDPexa-Vec), Arbovirus (e.g., VSV-GFP Indiana serotype, VSVgm-icv), and Paramyxovirus (e.g., MV-eGFP (Edmonston strain)).
[0383] Embodiment 356: The method according to any one of embodiments 337-
355, wherein said cancer cells in which immunogenic cell death is induced are characterized by elevated calreticulin (CRT) expression as compared to the same cells in which ICD is not induced. [0384] Embodiment 357: The method according to any one of embodiments 337-
356, wherein said cancer cells in which immunogenic cell death is induced are characterized by elevated expression and/or release of HMGB1 and/or elevated ATP release as compared to the same cells in which ICD is not induced. [0385] Embodiment 358: The method according to any one of embodiments 337-
357, wherein said cancer cells in which immunogenic cell death (ICD) is induced are of the same type of cancer that is to be treated or prevented.
[0386] Embodiment 359: The method according to any one of embodiments 337-
358, wherein the cancer to be treated or prevented is a cancer selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), acute lymphoblastic leukemia
(ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi sarcoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, kaposi sarcoma, kidney cancer (e.g., renal cell, Wilm's tumor, and other kidney tumors), langerhans cell histiocytosis, laryngeal cancer, leukemia, acute
lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myelogenous (CML), hairy cell, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer {e.g., childhood, non-small cell, small cell), lymphoma {e.g., AIDS-related, Burkitt {e.g., non-Hodgkin lymphoma), cutaneous T-Cell {e.g., mycosis fungoides, Sezary syndrome), Hodgkin, non-Hodgkin, primary central nervous system (CNS)), macroglobulinemia, Waldenstrom, male breast cancer, malignant fibrous
histiocytoma of bone and osteosarcoma, melanoma {e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple
myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes,
Myelogenous Leukemia, Chronic (CML), multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma {e.g., Ewing, Kaposi, osteosarcoma, rhadomyosarcoma, soft tissue, uterine), Sezary syndrome, skin cancer {e.g., melanoma, Merkel cell carcinoma, basal cell carcinoma, nonmelanoma), small intestine cancer, squamous cell carcinoma, squamous neck cancer with occult primary, stomach (gastric) cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom
macroglobulinemia, and Wilm's tumor. [0387] Embodiment 360: The method according to any one of embodiments 337-
358, wherein the cancer to be treated or prevented is selected from the group consisting of pancreatic cancer, lung cancer, breast cancer, and colon cancer.
[0388] Embodiment 361 : The method according to any one of embodiments 337-
358, wherein the cancer to be treated or prevented is pancreatic ductal adenocarcinoma (PDAC).
[0389] Embodiment 362: The method according to any one of embodiments 337-
361, wherein said cancer cells in which immunogenic cell death is induced are obtained from a tumor. [0390] Embodiment 363 : The method according to any one of embodiments 337-
362, wherein said cancer cells in which immunogenic cell death is induced are obtained from a primary tumor.
[0391] Embodiment 364: The method according to any one of embodiments 337- 363, wherein said cancer cells in which immunogenic cell death is induced are obtained from a tumor in the mammal that is to be treated.
[0392] Embodiment 365: The method according to any one of embodiments 337-
364, wherein said cancer cells in which immunogenic cell death is induced are obtained from a tumor biopsy, bloodstream, peritoneal fluid, pleural fluid, synovial fluid or bone marrow aspirate.
[0393] Embodiment 366: The method according to any one of embodiments 337-
364, wherein said cancer cells in which immunogenic cell death is induced are obtained from a resected primary or metastatic tumor.
[0394] Embodiment 367: The method according to any one of embodiments 337- 366, wherein said treatment is performed in the absence of surgical intervention or prior to surgical intervention.
[0395] Embodiment 368: The method according to any one of embodiments 337-
366, wherein said treatment is performed after surgical resection of a tumor or fine need biopsy. [0396] Embodiment 369: The method according to any one of embodiments 337-
368, wherein said treatment performed in conjunction with radiotherapy, proton beam therapy or photodynamic therapy.
[0397] Embodiment 370: The method according to any one of embodiments 337-
369, wherein said mammal is one in which a cancer or malignancy is diagnosed. [0398] Embodiment 371 : The method according to any one of embodiments 337-
362, wherein said mammal is treated prophylactically in the absence of a cancer diagnosis.
[0399] Embodiment 372: The method of embodiment 371, wherein said mammal is a mammal with a family history of cancer.
[0400] Embodiment 373 : The method according to any one of embodiments 371- 372, wherein said mammal has a genetic marker for elevated cancer risk. [0401] Embodiment 374: The method according to any one of embodiments 371-
373, wherein said mammal has mutation in Kras, BRCA1, BRCA2, and/or p53.
[0402] Embodiment 375 : The method according to any one of embodiments 337-
374, wherein said cancer cells in which immunogenic cell death (ICD) is induced are from a cancer or immortalized cell line.
[0403] Embodiment 376: The method of embodiment 375, wherein said cancer cells in which immunogenic cell death (ICD) is induced are from a cancer cell line or
immortalized cell line shown in Table 1.
[0404] Embodiment 377: The method according to any one of embodiments 337- 376, wherein said vaccination comprises cutaneous and/or subcutaneous vaccination.
[0405] Embodiment 378: The method according to any one of embodiments 337-
376, wherein said vaccination comprises intramuscular vaccination.
[0406] Embodiment 379: The method according to any one of embodiments 337-
378, wherein said vaccination is without an additional adjuvant. [0407] Embodiment 380: The method according to any one of embodiments 337-
378, wherein said vaccination is with an additional adjuvant.
[0408] Embodiment 381 : The method of embodiment 380, wherein said adjuvant is selected from the group consisting of alum, Squalene-Oil-in-water (e.g., MF59®), a PRR ligand, TLR3 and RLR Ligands, TLR4 ligands, TLR5 ligands, TLR7/8 ligands, TLR9 ligands, NOD2 ligands, and RLP3 inflammasome activators.
[0409] Embodiment 382: The method according to any one of embodiments 337-
381, wherein said mammal is a human.
[0410] Embodiment 383 : The method according to any one of embodiments 337-
381, wherein said mammal is a non-human mammal. [0411] Embodiment 384: A kit for the treatment or prophylaxis of a cancer said kit comprising:
[0412] a container containing an IDO pathway inhibitor; and/or
[0413] a container containing an agent that induces immunogenic cell death (ICD) (ICD-inducer); and/or
[0414] nanovesicle drug carriers according to any one of embodiments 1-59;
[0415] and/or nanoparticle drug carriers according to any one of embodiments 132-195; and/or
[0416] nanomaterial carriers according to any one of embodiments 196-254.
[0417] Embodiment 385 : The kit of embodiment 384, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l-methyl-tiyptophan (L-1MT), a mixture of D-1MT and L-1MT, 1- methyl-L-tryptophan (L-1MT), methylthiohydantoin-dl -tryptophan (MTH-Trp, Necrostatin), β-carbolines (e.g., 3-butyl-P-carboline), Naphthoquinone-based (e.g., annulin-B), S-allyl- brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2- (benzo[b]thiophen-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl- dithiocarbamate, S-hexyl-brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2- (indol-3-yl)ethyl]-S[(naphth-2-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid- 3-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-4-yl)methyl]- dithiocarbamate, 5-bromo-brassinin, Phenylimidazole-based IDO inhibitors (e.g., 4- phenylimidazole), Exiguamine A, imidodicarbonimidic diamide,N-methyl-N'-9- phenanthrenyl-monohydrochloride (NSC401366), INCB024360 (Epacadostat), 1- cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5-yl)ethanol (GDC-0919), IDO 1 -derived peptide, NLG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl- 2-thiopseudourea hydrochloride, and 4-phenylimidazole.
[0418] Embodiment 386: The kit of embodiment 384, wherein said IDO inhibitor comprises indoximod.
[0419] Embodiment 387: The kit of embodiment 384, wherein said IDO inhibitor comprise an IDO inhibitor conjugated to a phospholipid according to any one of
embodiments 103-131.
[0420] Embodiment 388: The kit according to any one of embodiments 384-387, wherein said ICD inducer comprises a chemotherapeutic agent selected from the group consisting of oxaliplatin, cisplatin, doxorubicin, epirubicin, idarubicin, mitoxantrone, anthracenedione, bleomycin, bortezomib, R2016, and cyclophosphamide.
[0421] Embodiment 389: The kit of embodiment 388, wherein said ICD inducer comprises oxaliplatin. [0422] Embodiment 390: The kit according to any one of embodiments 384-389, wherein said ICD inducer comprises an oncolytic virus. [0423] Embodiment 391 : The kit of embodiment 390, wherein said oncolytic virus is selected from the group consisting of Parvovirus (e.g., H-PV), Adenovirus (e.g., hTERT-Ad, Ad5/3-D24-GMCSF), Herpes simplex virus (e.g., G207, HSV-1716, T-VEC, HSV-2 ΔΡΚ mutant), Poxvirus (e.g., vSP, vvDDPexa-Vec), Arbovirus (e.g., VSV-GFP Indiana serotype, VSVgm-icv), and Paramyxovirus (e.g., MV-eGFP (Edmonston strain)).
[0424] Embodiment 392: The kit according to any one of embodiments 384-391, wherein said kit contains both an IDO inhibitor and an ICD inducer.
[0425] Embodiment 393 : The kit of embodiment 392, wherein said IDO inhibitor and said ICD inducer are in separate containers. [0426] Embodiment 394: The kit of embodiment 392, wherein said IDO inhibitor and said ICD inducer are in the same container.
[0427] Embodiment 395 : The kit of embodiment 394, wherein said IDO inhibitor and said ICD inducer are provided as a nanoparticle drug carrier according to any one of embodiments 132-195. [0428] Embodiment 396: A formulation for inducing immunogenic cell death, said formulation comprising a nanomaterial that induces ICD.
[0429] Embodiment 397: The formulation of embodiment 396, wherein said formulation comprises a pharmaceutical formulation comprising said nanomaterial that induces ICD and a pharmaceutically acceptable carrier. [0430] Embodiment 398: The formulation according to any one of embodiments
396-397, wherein said formulation is a unit dosage formulation.
[0431] Embodiment 399: The formulation according to any one of embodiments
396-398, wherein said formulation is sterile.
[0432] Embodiment 400: The formulation according to any one of embodiments 396-399, wherein said nanomaterial contains or comprises a nanomaterial that induces ICD.
[0433] Embodiment 401 : The formulation of embodiment 400, wherein said nanomaterial that induces ICD forms a nanoparticle.
[0434] Embodiment 402: The formulation of embodiment 400, wherein said nanomaterial that induces ICD comprises a core-shell nanoparticle. [0435] Embodiment 403 : The formulation of embodiment 400, wherein said nanomaterial that induces ICD comprises a doped nanoparticle.
[0436] Embodiment 404: The formulation according to any one of embodiments
396-403, wherein said nanomaterial comprises a material selected from the group consisting of CuO, Cu20, Sb203, As2C"3, Bi203, P203, ZnO, Ti02, graphene oxide, and 2D materials other than graphene or graphene oxide.
[0437] Embodiment 405 : The formulation of embodiment 404, wherein said nanomaterial comprises CuO.
[0438] Embodiment 406: The formulation according to any one of embodiments 404-405, wherein said nanomaterial comprises Sb203.
[0439] Embodiment 407: The formulation according to any one of embodiments
404-406, wherein said nanomaterial comprises ZnO.
[0440] Embodiment 408: The formulation according to any one of embodiments
404-407, wherein said nanomaterial comprises Ti02. [0441] Embodiment 409: The formulation according to any one of embodiments
404-408, wherein said material comprise a 2D material.
[0442] Embodiment 410: The formulation of embodiment 409, wherein said 2D material comprises a material selected from the group consisting of graphene, graphyne, borophene, germanene, silicene, Si2BN, stanene, phosphorene, bismuthene, molybdenite, metals, and a 2D supracrystal.
[0443] Embodiment 41 1 : The formulation of embodiment 410, wherein said nanomaterial comprises graphene oxide.
[0444] In certain embodiments the agent(s) that induce ICD exclude cisplatin, and/or in certain embodiments the agent(s) that induce ICD exclude doxorubicin. DEFINITIONS
[0445] The terms "subject," "individual," and "patient" may be used interchangeably and refer to humans, as well as non-human mammals (e.g., non-human primates, canines, equines, felines, porcines, bovines, ungulates, lagomorphs, and the like). In various embodiments, the subject can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, as an outpatient, or other clinical context. In certain embodiments, the subject may not be under the care or prescription of a physician or other health worker.
[0446] As used herein, the phrase "a subject in need thereof refers to a subject, as described infra, that suffers from, or is at risk for a cancer as described herein. Thus, for example, in certain embodiments the subject is a subject with a cancer {e.g., pancreatic ductal adenocarcinoma (PDAC), breast cancer {e.g., drug-resistant breast cancer), colon cancer, brain cancer, and the like). In certain embodiments the methods described herein are prophylactic and the subject is one in whom a cancer is to be inhibited or prevented. In certain embodiments the subject for prophylaxis is one with a family history of cancer and/or a risk factor for a cancer {e.g., a genetic risk factor, an environmental exposure, and the like).
[0447] The term "treat" when used with reference to treating, e.g., a pathology or disease refers to the mitigation and/or elimination of one or more symptoms of that pathology or disease, and/or a delay in the progression and/or a reduction in the rate of onset or severity of one or more symptoms of that pathology or disease, and/or the prevention of that pathology or disease. The term treat can refer to prophylactic treatment which includes a delay in the onset or the prevention of the onset of a pathology or disease.
[0448] The terms "coadministration" or " administration in conjunction with" or
"cotreatment" when used in reference to the coadministration of a first compound (or component) {e.g., an ICD inducer) and a second compound (or component) {e.g., an IDO inhibitor) indicates that the first compound (or component) and the second compound (or component) are administered so that there is at least some chronological overlap in the biological activity of first compound and the second compound in the organism to which they are administered. Coadministration can include simultaneous administration or sequential administration. In sequential administration there may even be some substantial delay {e.g., minutes or even hours) between administration of the first compound and the second compound as long as their biological activities overlap. In certain embodiments, the coadminstration is over a time frame that permits the first compound and second compound to produce an enhanced therapeutic or prophylactic effect on the organism. In certain embodiments the enhanced effect is a synergistic effect. [0449] The term "immunogenic cell death" or "ICD" refers to a unique form of cell death caused by some cytostatic agents such as anthracyclines (Obeid et al. (2007) Nature Med., 13(1): 54-61), oxaliplatin and bortezomib, or radiotherapy and photodynamic therapy (PDT). Unlike regular apoptosis, which is mostly non-immunogenic or even tolerogenic, immunogenic apoptosis of cancer cells can induce an effective antitumor immune response through activation of dendritic cells (DCs) and consequent activation of specific T cell response (Spisek and Dhodapkar (2007) Cell Cycle, 6(16): 1962-1965). Endoplasmic reticulum (ER) stress, reactive oxygen species (ROS) production and induction of autophagy are key intracellular response pathways that govern ICD (Krysko et al. (2012) Nat. Rev. Cane. 12(12): 860-875). In addition to facilitating tumor cell death that facilitates antigen presentation by dendritic cells, ICD is characterized by secretion or release of damage- associated molecular patterns (DAMPs), which exert additional immune adjuvant effects. Calreticulin (CRT), one of the DAMP molecules, which is normally in the lumen of the ER, is translocated to the surface of dying cell where it functions as an "eat me" signal for phagocytes. Other important surface exposed DAMPs are heat-shock proteins (HSPs), namely HSP70 and HSP90, which under stress condition are also translocated to the plasma membrane. On the cell surface they have an immunostimulatory effect, based on their interaction with number of antigen-presenting cell (APC) surface receptors like CD91 and CD40 and also facilitate cross-presentation of antigens derived from tumor cells on MHC class I molecule, which then triggers CD8+ T cell activation and expansion. Other important DAMPs, characteristic for ICD are secreted amphoterin (HMGB 1) and ATP {see, e.g., Apetoh et a/. (2007) Nature Med. 13(9): 1050-1059; Ghiringhelli et al. (2009) Nature Med. 15(10): 1170-1178). HMGB 1 is considered to be a late apoptotic marker and its release to the extracellular space appears to be required for the optimal release and presentation of tumor antigens to dendritic cells. It binds to several pattern recognition receptors (PRRs) such as Toll-like receptor (TLR) 2 and 4, which are expressed on APCs. The most recently found DAMP released during immunogenic cell death is ATP, which functions as a "find- me" signal for monocytes when secreted and induces their attraction to the site of apoptosis (see, e.g., Garg et al. (2012) EMBO J. 31(5): 1062-1079). ATP binds to purinergic receptors on APCs.
[0450] The terms "IDO inhibitor", "IDO pathway inhibitor", and "inhibitor of the IDO pathway) are used interchangeably and refer to an agent (a molecule or a composition) that either partially or fully blocks the activity of indoleamine-2,3-dioxygenase (IDO) and/or partially or fully suppresses the post-enzymatic signaling cascade(s) in the IDO pathway. IDO is an intracellular heme-containing enzyme that initiates the first and rate-limiting step of tryptophan degradation along the kynurenine pathway. The indoleamine 2, 3 -di oxygenase (IDO) pathway regulates immune response by suppressing cytotoxic T cell function, enhancing regulatory T cell activity (Tregs) and enabling tumor immune escape, either at the tumor or regional lympnode sites. An IDO pathway inhibitor can inhibit the IDO enzyme directly or by interfering or perturbing IDO effector pathway components. Such components include, but are not limited to: ID02, tryptophan 2,3-dioxygenase (TDO), the mammalian target of rapamycin (mTOR) pathway, arylhydrocarbon receptor (AhR) pathway, the general control nonderepressible 2 (GCN2) pathway, and the AhR/IL-6 autocrine loop.
[0451] The terms "nanocarrier" and "nanoparticle drug carrier" are used
interchangeably and refer to a nanostructure having a porous interior core (e.g., a "porous nanoparticle"). In certain embodiments the nanocarrier comprises a lipid bilayer encasing (or surrounding or enveloping) the porous particle core. In certain embodiments the nanoparticle is a porous silica nanoparticle (e.g., mesoporous silica nanoparticle or "MSNP").
[0452] As used herein, the term "lipid" refers to conventional lipids, phospholipids, cholesterol, chemically functionalized lipids for attachment of PEG and ligands, etc. [0453] As used herein, the terms "lipid bilayer" or "LB" refers to any double layer of oriented amphipathic lipid molecules in which the hydrocarbon tails face inward to form a continuous non-polar phase.
[0454] As used herein, the terms "liposome" or "lipid vesicle" or "vesicle" are used interchangeably to refer to an aqueous compartment enclosed by a lipid bilayer, as being conventionally defined (see, e.g., Stryer (1981) Biochemistry, 2d Edition, W. H. Freeman & Co., p. 213).
[0455] A "nanovesicle" refers to a "lipid vesicle" having a diameter (or population of vesicles having a mean diameter) ranging from about 20 nm, or from about 30 nm, or from about 40 nm, or from about 50 nm up to about 500 nm, or up to about 400 nm, or up to about 300 nm, or up to about 200 nm, or up to about 150 nm, or up to about 100 nm, or up to about 80 nm. In certain embodiments a nanovesicle has a diameter ranging from about 40 nm up to about 80 nm, or from about 50 nm up to about 70 nm.
[0456] Compared with the lipid bilayer coated on mesoporous silica nanopaticles, the lipid bilayer in a lipid vesicle or liposome can be referred to as an "unsupported lipid bilayer" and the lipid vesicle itself (when unloaded) can be referred to as an "empty vesicle". The lipid bilayer coated on mesoporous silica nanopaticles can be referred to as a "supported lipid bilayer" because the lipid bilayer is located on the surface and supported by a porous particle core. In certain embodiments, the lipid bilayer can have a thickness ranging from about 6 nm to about 7 nm which includes a 3-4 nm thickness of the hydrophobic core, plus the hydrated hydrophilic head group layers (each about 0.9 nm) plus two partially hydrated regions of about 0.3 nm each. In various embodiments, the lipid bilayer surrounding the silica nanoparticle comprises a continuous bilayer or substantially continuous bilayer that effectively encapsulates and seals the nanoparticle.
[0457] As used herein, the term "selective targeting" or "specific binding" refers to use of targeting ligands on the surface of a drug delivery nanocamer (e.g., a LB-coated nanoparticle). In certain embodiments the targeting ligand(s) are on the the surface of a lipid bilayer of LB-coated nanoparticle. Typically, the ligands interact specifically/selectively with receptors or other biomolecular components expressed on the target, e.g., a cell surface of interest. The targeting ligands can include such molecules and/or materials as peptides, antibodies, aptamers, targeting peptides, polysaccharides, and the like.
[0458] A coated mesoporous silica nanopaticle, having targeting ligands can be referred to as a "targeted nanoparticle or a targeted drug delivery nanocarrier (e.g., LB-coated nanoparticle).
[0459] The term "about" or "approximately" as used herein refers to being within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. the limitations of the measurement system, i.e. the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5% and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term "about" meaning within an acceptable error range for the particular value should be assumed.
[0460] The term "drug" as used herein refers to a chemical entity of varying molecular size, small and large, naturally occurring or synthetic, that exhibits a therapeutic effect in animals and humans. A drug may include, but is not limited to, an organic molecule (e.g., a small organic molecule), a therapeutic protein, peptide, antigen, or other biomolecule, an oligonucleotide, an siRNA, a construct encoding CRISPR cas9 components and, optionally one or more guide RNAs, and the like.
[0461] A "pharmaceutically acceptable carrier" as used herein is defined as any of the standard pharmaceutically acceptable carriers. The pharmaceutical compositions of the subject invention can be formulated according to known methods for preparing
pharmaceutically useful compositions. The pharmaceutically acceptable carrier can include diluents, adjuvants, and vehicles, as well as carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating material that does not react with the active ingredients of the invention. Examples include, but are not limited to: phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions. The carrier can be a solvent or dispersing medium containing, for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Formulations are described in a number of sources that are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Sciences (Martin E W [1995] Easton Pa., Mack Publishing Company, 19th ed.) describes formulations which can be used in connection with the drug delivery nanocarrier(s) (e.g., LB-coated nanoparticle(s)) described herein.
[0462] As used herein, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of
immunoglobulin genes or derived therefrom that is capable of binding (e.g., specifically binding) to a target (e.g., to a target polypeptide). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
[0463] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. [0464] Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chainjoined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region {see, Fundamental Immunology, W E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Certain preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), more preferably single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. The single chain Fv antibody is a covalently linked VH-VL heterodimer which may be expressed from a nucleic acid including VH- and VL- encoding sequences either joined directly or joined by a peptide-encoding linker. Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. While the VH and VL are connected to each as a single
polypeptide chain, the VH and VL domains associate non-covalently. The first functional antibody molecules to be expressed on the surface of filamentous phage were single-chain Fv's (scFv), however, alternative expression strategies have also been successful. For example Fab molecules can be displayed on a phage if one of the chains (heavy or light) is fused to g3 capsid protein and the complementary chain exported to the periplasm as a soluble molecule. The two chains can be encoded on the same or on different replicons; the important point is that the two antibody chains in each Fab molecule assemble post- translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to, e.g., g3p (see, e.g., U.S. Patent No: 5733743). The scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three- dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (see e.g., U.S. Patent Nos. 5,091,513, 5, 132,405, and 4,956,778). In certain embodiments antibodies should include all that have been displayed on phage (e.g., scFv, Fv, Fab and disulfide linked Fv (see, e.g, Reiter et al. (1995) Protein Eng. 8: 1323-1331) as well as affibodies, unibodies, and the like.
[0465] The term "specifically binds", as used herein, when referring to a biomolecule
(e.g., protein, nucleic acid, antibody, etc.), refers to a binding reaction that is determinative of the presence of a biomolecule in heterogeneous population of molecules (e.g., proteins and other biologies). Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody or stringent hybridization conditions in the case of a nucleic acid), the specified ligand or antibody binds to its particular "target" molecule and does not bind in a significant amount to other molecules present in the sample. [0466] "Two-dimensional materials (2D materials) are materials that do not require a substrate to exist. In other words, they can be isolated as freestanding one atom thick sheets. As a practical matter, this definition can be relaxed to include materials with a thickness of a few atoms (e.g., less than about 10 atoms).
[0467] The term "substantially pure isomer" refers to a formulation or composition wherein among various isomers of a compound a single isomer is present at 70%, or greater or at 80% or greater, or at 90% or greater, or at 95% or greater, or at 98% or greater, or at 99%) or greater, or said compound or composition comprises only a single isomer of the compound.
[0468] A "bioreactive nanomaterial" refers to an engineered biomaterial that induces or catalyzes a biological response. In certain embodiments the nanomaterial induces a response by virtue of one or more properties selected from the group consisting of composition, size, shape, aspect ratio, dissolution, electronic, redox, surface display, surface coating, hydrophobic, hydrophilic, an atomically thin nanosheet, or functionalized surface groups" to catalyze the biological response at various nano/bio interfaces. In certain embodiments the bioreactive nanomaterial has the ability to induce ICD biological responses in cells (e.g., in tumor cells) and/or as well as activating the innate immune system through delivery of "danger signal" and adjuvant effects.
BRIEF DESCRIPTION OF THE DRAWINGS
[0469] Figure 1 provides a schematic explaining immunogenic cell death and synergy with an IDO inhibitor (indoximod). DOX delivery to the primary BC site induce a form of stress-induced cell death, characterized by as CRT expression (an "eat-me" signal for dendritic cell uptake), as well as the release of the adjuvant stimuli, HMGB-1 and ATP. The ability of maturating dendritic cells to cross-present endogenous tumor antigens to naive CD8+ T-cells, induces the development of cytotoxic T-cells. CTLs are capable of inducing perforin and granzyme B induced death of primary and metastatic cancer cells. The co- delivery of indoximod enhances the ICD effect (increased autophagy and ATP production), in addition to interfering in the metabolic immunosuppressive IDO pathway. This induces a synergistic innate and cognitive immune response at the cancer site, and also assists long- lived memory T cell development.
[0470] Figure 2 illustrates the structure of indoximod and various other IDO pathway inhibitors. [0471] Figure 3 illustrates representative examples to show the use of an ester bond to make IDO inhibitor (e.g., indoximod) pro-drug conjugates. As a general strategy, the H2 group (highlighted by red circle) in the indoximod is protected before the conjugation reaction. The -COOH (green box) in indoximod can then robustly react with the -OH (blue box) in PHGP, Vitamin E or cholesterol, leading to a list of pro-drugs, that can self-assemble as vesicles (or micellar structures) in aqueous solution. It can also be used in the lipid mixture for MS P coating. We have now also made the L-isomer successfully by using the same ester bond and have characterized the product by the same sophisticated proton NMR to actually show that we can make the product (see, e.g., Example 6).
[0472] Figure 4 illustrates representative examples to show the combined use of HO-
Figure imgf000065_0001
linker and ester bond to make IDO inhibitor (e.g., indoximod) pro-drug conjugates. As illustrated in this example, the NH2 group (highlighted by red circle) is protected in the indoximod before the conjugation reaction. The -COOH (green box) in indoximod can robustly react with one -OH group (blue box) in the linker compound, which can also readily react with -COOH in the oleic acid or DHA molecule via the other -OH group. We have also made the L-isomer successfully by using the same ester bond and have characterized the product by the same proton NMR (see, e.g., Example 6).
[0473] Figure 5 A shows construction of an IND nanovesicle by self-assembly of
IND-PL. Figure 5B whows CryoEM images of DOX/IND-NVs versus commercial DOXIL® liposome. [0474] Figure 6 illustrates the synthesis of DOX-laden IND-PL coated MSNP.
[0475] Figure 7 illustrates western blot assays showing the enhanced effect of IND-
NV on mTOR signaling. [0476] Figures 8A-8D illustrate a biodistribution study in a 4T1 orthotopic model.
Fig. 8A), IVIS imaging to look at primary tumor burden by bioluminescence. Fig. 8B shows the same model shown in Fig. 8 A after receiving IV injection of free Dox, DOXIL® and Dox/IND-NV at identical Dox dose (5mg/kg). Fig. 8C shows results of a separate PK study in which a single IV injection of free Dox, DOXIL® or Dox/IND-NV (Dox 5 mg/kg) was carried out. Fig. 8D shows indoximod concentration measured using UPLC.
[0477] Figures 9 A and 9B shows ongoing anti-cancer efficacy data in 4T1 orthotopic breast cancer bearing mice. Figure 9A: One million 4Tl-luc cells were injected into the mammary fat pad of Balb/c mice at day 0. The treatments were launched when the tumor size reached around -100 mm3 (day 8). Up to the date shown, a total of 4 IV injections of Dox/IND-NV (Dox 5 mg/kg; IND-PL 31.5 mg/kg; molar ratio of DOX:IND-PL=l :5) were performed (indicated by arrow). Controls include saline, free Dox, DOXIL® liposome and free Dox + IND-NV at identical drug doses. Tumor size was measured 2-3 times per week. While the animal efficacy experiment is still ongoing, a promising anticancer trend already emerges in mice receiving Dox/IND-NV. Fig. 9B: In fact, in the experiment shown in (Fig. 9A), we also included additional treatment using free Dox plus anti-PDl with a view to demonstrate the advantage of Dox/IND-NV versus a standard chemo/immuno combination therapy in breast cancer.
[0478] Figure 10, panels A-C, illustrates the use of a vaccination approach to identify ICD inducers. Consensus guidelines were used to identify DOX and PTX as ICD introducing chemo in vitro and in vivo. Panel A: CRT surface expression was detected flow cytometry, using the indicated drugs at different doses over 24 hr. Screening for FDVIGBl and ATP yielded similar results. Panel B: Animal vaccination study using 2 rounds of subcutaneous injection of dying 4T1 cells one week apart, followed by injecting live cells SC on the contralateral side. Panel C: Spaghetti curves showing interference in 4T1 tumor growth by vaccinating with cells undergoing ICD by DOX and PTX.
[0479] Figure 11, panels A-D, illustrates the synthesis, characterization and PK assessment of a DOX/IOND liposome (vesicle). Panel A depicts synthesis of the DOX and IND-PL liposome. Schematic preparation procedures that pinpoints each steps utilized for coining the DOX/IND-Liposome via remote loading approach. Briefly, a lipid film comprising IND-PL, Cholesterol, and DSPE-PEG2K was obtainned by removing the organic solvent using rotary evaporator, which was hydrated in protonating agent, (NH4)2S04 followed by active DOX remote loading. The box in panel A summarizes the IND-PL self- assembly and active DOX loading. This begins by conjugating IND to a phospholipid to yield the prodrug, IND-PL. The prodrug is amphipathic, allowing self-assembly into liposomes in aqueous buffer. The entrapment of ammonium sulfate at the time of assembly, permits or DOX import into the liposome by a proton gradient. Panel B: Cartoon showing the self-assembly of DOX-laden IND-Liposome; high magnification cryoEM shows the clear bilayer structure with a chunk of (DOX- H3)2S04 precipitate presented in the empty core. Neutral DOX, is a weak basic molecule that can diffuse across the IND-PL lipid bilayer to the empty inner core of IND-Liposome, where it met with the released free protons NH4+, and S04 2" from prepackaged (NH4)2S04, which converted DOX to (DOX-NH3)2S04 precipitate. Upon formation of precipitate, DOX was unable to cross back the lipid bilayer. Panel C: Side by side comparison of DOX/IND-Liposome and DOXIL® in terms of DOX and IND loading, size, polydispersity, charge and the endotoxin level. Panel D: DOX precipitates as crystals in the nanovesicle, with identical morphology to DOXIL® as shown in the CryoEM picture. DLS sizes of DOX/IND-liposome is very similar to DOXIL®. [0480] Figure 12, panels A-E, illustrates the biodistribution and PK assessment of a
DOX/IND liposome. DOX/IND-Liposome achieved comparable pharmacokinetics (PK) and tumor uptake as DOXIL®, which are much improved compared to free DOX. Panel A: Establishment of syngeneic 4T1 orthotopic model that will be used for PK, biodistribution and efficacy study. Autopsy and IVIS images of the 4T1 -derived orthotopic BC model in immunocompetent Balb/c mice. Briefly, luciferase-transfected 4T1 BC cells (1 million in 1/1, v/v, matrigel/DMEM) were injected to the 2nd mammary fat pad of Balb/c mice. Two weeks post 4T1 cells inoculation, a visible tumor mass was observed with detectable lung metastasis; 4 weeks later, a big tumor chunk was discerned with severe lung metastasis. Panel B: DOX MTD and rationale for choosing the proper and clinically relevant DOX dose. Following the NCI protocol, the MTD for DOX, DOXIL®, and DOX/IND-Liposome were determined at 8, 15, and 15 mg/kg for respectively. Clinically, DOXIL® is IV administered at a dose of -50 mg/cm2 once a month. This allows us to use this formula to convert the human dose into mouse dose. A human DOX dose of 50 mg/cm2/month equals to DOX mouse dose of -16.4 mg/kg per month. Consider the MTD of free DOX and frequency to be used; the safe DOX dosing regimen is at 5 mg/kg for 3 times via IV injection within one month period in mice. Panel C: Biodistribution of DOX fluorescence in 4T1 orthotopic tumor model IV injected with free DOX, DOXIL®, and DOX/IND-Liposome at 5 mg/kg DOX dose (n=3). Mice were sacrificed 24 h post IV injection, and imaged by IVIS imaging (excitation filter: 500 nm and Emission filter: DsRed), and quantified by LIVING IMAGE® software (PerkinElmer, version 4.5) (*P < 0.01, ANOVA). Panels D and E: PK and drug tissue distribution in 4T1 orthotopic tumor model IV injected with free DOX, DOXIL®, DOX/IND-Liposome, and/or free IND at equivalent DOX dose 5 mg/kg (n=6). The blood circulation curve for different DOX (panel D) or IND formulations (panel E) in % injected drug dose (left), and % injected drug/g tissue (right). Results are expressed as mean ± SEM. **p < 0.01, (ANOVA).
[0481] Figure 13 illustrates the treatments used in the 4T1 orthotopic breast cancer
(BC) model described in Example 3 (see also Table 8 therein). [0482] Figure 14, panels A-E, shows that DOX/IND-Liposome outperforms
DOXIL® in restraining the orthotopic BC in immunocompetent mice. Panel A: DOX/IND- Liposome (#7, DOX: 5 mg/kg, IND dose was at 8.7 mg/kg) was IV injected to mice on days 8, 11, and 14, respectively. Controls including Saline (#1), DOX (#2), DOXIL® (#3), IND- Liposome (#4), and DOX + IND-Liposome (#5), received identical dose of DOX or IND via IV. Tumor growth inhibition curve, showed markedly improved antitumor efficacy using DOX/IND liposome versus controls (**P < 0.01, ANOVA). IHC showing IDO expression (brown color) at the orthotopic tumor site. Panels B and C: Representative tumor images and averaged tumor weights from different groups after sacrificing mice on day 22. Panel D: Representative lung metastasis from each group. The quantified bioluminescence intensity displays the drastically reduced tumor lung metastasis in #1 as compared to #3 and other controls (**P < 0.01, ANOVA), notwithstanding #3 minimized the lung metastasis when compared to other controls (*P < 0.05, ANOVA). Panel E: Dual delivery #7, dramatically prolonged the mice survival as manifested by the Kaplan-Meier analysis (**P < 0.01, Log- rank Mantel-Cox test). [0483] Figure 15, panels A-F, shows that combined use of anti-PD-1 immune checkpoint inhibitor with DOX/IND-Liposome led to the further boosted tumor control with completely depleted metastasis. Panel A: IHC staining showing the high PD-1 expression in the 4T1 BC tissue, which provides a legitimate reason for combining anti PD-1 therapy. Panel B: Tumor volume curve over the time post different treatments, in which combination of anti PD-1 (IP injected at 100 μg/mouse on day 8, 11, and 14) and DOX/IND-Liposome (# 8) furthered the tumor suppression significantly. Panels C and D: Selective dissected tumors from the mice in #1, #6, #7, and #8, and their calculated averaged tumor weights. Panel E: Representative lung metastasis images from #1, #6, #7, and #8. Combination of anti PD-1 and DOX/IND-Liposome resulted in complete lung metastasis remission. Panel F: The mice survival was further lengthened in #8. Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01, (ANOVA).
[0484] Figure 16, panels A-D, shows that systemic knockdown of CD8 T cells drastically reduced the antitumor efficacy of DOX/IND-Liposome in 4T1 BC mice model. To elucidate whether the improved tumor reduction by DOX/IND-Liposome was attributed to the stimulated cytotoxic CD8 T lymphocytes arisen from the combination of ICD and IDO inhibition. CD8+ T cells were systemically depleted by IP injecting anti CD8 3 days before the first drug treatments and were continued every 2-3 days until the termination of the study. Panel A: Upon injecting the anti-CD8 antibody, the tumor growth delay from DOX/IND- Liposome were prominently decreased. Panel B: IVIS imaging showing the representative bioluminescence of the orthotopic 4T1 tumor development, which aligns with the tumor growth curve exhibited in A. Panel C: Representative lung metastasis images from #1, #1 and #9, where systemic knockdown of CD8 led to the recovery of lung metastasis. Panel D: CD8 depletion also contributed to the drastically lessened mice survival rate. Results are expressed as mean ± SEM. **p < 0.01, (ANOVA).
[0485] Figure 17, panels A-F, shows that the DOX/IND-liposome antitumor effect involves the activation of both innate and adaptive immunity in breast cancer. A panel of immune biomarkers was comprehensively evaluated in the tumors from the orthotopic 4T1 tumor model shown in Table 13 (Example 3). Panel A: Multi-color flow cytometry analysis for adaptive immune markers: CD8 (CD45+CD3+CD8+CD25+)/Treg
(CD45+CD3+CD4+Foxp3+) ratios, IFN-y+ T cells (CD45+CD3+CD8+IFN-Y+), and Granzyme B+ T cells (CD45+CD3+CD8+Granzyme B+). Panel B: Multi-color flow cytometry analysis for innate immune markers: CD91+DC-like cells (CD45+CD1 lb+CDl lc+CD91+),
CD80+/CD86+ DCs (CD45+CD1 lc+CD80+CD86+), and CD103+ DCs
(CD45+CD1 lb+CDl lc+CD103+). Dual delivery #7 significantly improved the CD8/Tregs ratios, IFN-y+ and Granzyme B+ T cells, and CD91+, CD80+/CD86+, and CD103+ DCs, particularly when combined with anti PD-1 immune checkpoint blockade. These
enhancements were revoked upon depleting CD8 T cells systemically. IHC staining for CD8 (panel C), CRT (panel D), and perforin (panel E), respectively, this is in harmony with the flow cytometry results. Panel F: Western blotting of P-S6K in tumors in the groups treated with IND-Liposome. Elevated P-S6K intensity indicates the enhanced mTOR stimulation (left). Real-time PCR for the analysis of IL-6 levels at the tumor tissues, where the decreased IL-6 expression suggests the GCN2 and/or IL-6/STAT3/AHR autocrine signaling loop have been hampered (right). Both increased PS6K and reduced IL-6 manifested that
immunosuppressive IDO pathway has been effectively controlled.
[0486] Figure 18, shows the results of a safety assessment of DOX/IND liposome in mice. Assessment of blood chemistry to reveal the safety of different DOX formulations. Free DOX-bearing groups had severe toxicity as evidenced by the greatly increased levels of cardiac troponin I, creatine kinase, ALT, AST, and creatinine, while this phenomenon was not seen in dual delivery #7, substantiating its superior safety feature in vivo. Results are expressed as mean ± SEM. *p <0.05; **p <0.01; #p <0.001, (ANOVA). [0487] Figure 19, panels A-D, illustrates in vitro characterization of DOX ICD profile in 4T1 breast cancer cell line. Panel A: Flow cytometry analysis to show the normalized CRT expression levels after translocation from endoplasmic to cell surface in 4T1 cells treated with PBS (Ctr), Cis, DOX, PTX and OX at various concentrations for 24 h. Panel B: Confocal microscopy depicting the surface induction of CRT, in the presence of Cis (100 μΜ), DOX (1 μΜ), PTX (1 μΜ) and OX (50 μΜ) for 24 h, respectively in 4T1 cells. The CRT, cell nuclei, and surface membrane and were detected by ALEXAFLUOR®647- conjugated anti-CRT, Hoechst 33342, Alexa Fluor® 488-Conjugated Wheat Germ
Agglutinin antibody staining, respectively. Scale bar is 20 μπι. In the same experiments as shown A, the supernatants were subject to the analysis of HMGB-1 (Panel C) or ATP (Panel D) assessment using an HMGB-1 ELISA kit (IBL International GmbH) or ATPlite 1-step Luminescence Assay Kit (PerkinElmer). Results are expressed as mean ± SEM. *p <0.05; **p <0.01; *p <0.001, (ANOVA).
[0488] Figure 20 illustrates results of a vaccination study using dying 4T1 tumor cells treated with PBS, 100 μΜ Cis, 50 μΜ OX and 1 μΜ DOX for 24 h in female Balb/c mice (n=6). Excised tumors from the mice from each group; averaged tumor weights were plotted (right). Bioluminescence visualization of 4T1 SC tumor development on day 10, 16, and 19 respectively. Mice body weight monitoring was provided.
[0489] Figure 21, panels A-C, illustrates results of a vaccination study using dying
4T1 tumor cells treated with PBS, 100 μΜ Cis, 50 μΜ OX and 1 μΜ DOX for 24 h in female Balb/c mice (n=6). Panel A: Multi-color flow cytometry of CD8+ T lymphocytes
(CD45+CD3+CD4+CD25+) to Foxp3+ Tregs (CD45+CD3+CD4+Foxp3+) ratio (left); IHC staining for infiltrated tumor CD8+ T cells and Foxp3+ Tregs. Flow and IHC data were consistent to each other, where tumor CD8+ T cells in ICD-inducing DOX and PTX were significantly augmented with reduced Foxp3+ Tregs. Panel B: Multi-color flow cytometry for tumor IFN-γ (CD45+CD3+CD8+IFN-Y+) and Granzyme B (CD45+CD3+CD8+Granzyme B+) (upper panel), and IHC of Peforin and CC-3 (lower panel). Markedly improved IFN-γ, Granzyme B, Peforin, and CC-3 corrobarated that DOX and PTX can stimulate the adaptive immunity. Panel C: Measurement of CD103 (CD45+CD1 lb+CDl lc+CD103+) and
CD80/CD86 (CD45+CD1 lc+CD80+CD86+) by flow cytometry (left), and IHC for IL12p70. These results demonstrated that during DOX-triggered ICD, innate antitumor immune response was remarkablely boosted compared to PBS and Cis-treated groups. Results are expressed as mean ± SEM. *p <0.05; **p <0.01; (ANOVA).
[0490] Figure 22, panels A-H, illustrates in vivo 4T1 othotopic breast tumor efficacy evaluation as shown in Figure 16. Panel A: "Spaghetti plots" detailing each individual mouse tumor growth from different treatment groups. Panel B: Representative tumor
bioluminescence images on day 8, 11, 14, 17, and 22, respectively. Panel C: Mice weight change monitoring during the animal experiments. Mice were sacrificed on day 22 and tumors were obtained for flow cytometry and IHC analysis (Figure 16). IHC staining analysis for tumoral Foxp3 (panel D), CD91 (panel E), CC-3 (panel F), IL12p70 (panel G), and LC-3 (panel H). Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01,
(ANOVA). Scale bar islOO μιη. [0491] Figure 23, panels A-C, illustrates results of a mechanistic investigation of
IND-PL in 4T1 cells. Panel A: Western blotting to determine the phosphorylated S6 kinase (P-S6K) levels in 4T1 cells after being dosed with IND or IND-Liposome at 10 μΜ and 50 μΜ, respectively for 3h in tryptophan-deficient DMEM. The IND-Liposome showed significantly enhanced P-S6K induction in a dose dependent fashion as compared to free IND treatment, which is indicative of its superior mTOR pathway stimulation. A quantitative data of the P-S6K bands using image J software was displayed in the right panel based on three independent repeats. Panel B: The supernatants from the cell culture in panel A were collected for the analysis of IL-6 release from 4T1 cells. A dose dependent reduction of IL-6 release was observed in free IND-treated group, which was further weakened by IND- Liposome, implying that the GCN2 and/or IL-6/STAT3/AHR autocrine signaling loop was preeminently hampered. Panel C: Intracellular internalization of IND-Liposome in 4T1 cells. Cells were challenged with free IND or IND-Liposome at equivalent 100 μg/mL IND dose for 4, 24, and 72 h, respectively. Cell suspensions were then collected for UPLC-MS/MS measurement for IND or IND-PL following our established protocol. The data delineate that IND-PL drastically improved intracellular uptake of IND with efficient and rapid release of IND. Results are expressed as mean ± SEM. *p <0.05; **p <0.01; #p O.001, (ANOVA).
[0492] Figure 24 shows a schematic to illustrate how contemporaneous delivery of OX and IND, including through the creative use of nanocarriers, could induce effective anti- PDAC immunity. We hypothesized that the induction of ICD by OX and interference in the IDO pathway by IND could synergize in generating an effective anti-tumor immune response, premised on enhanced tumor cell uptake and antigen presentation by participating DCs, coupled with interference in the immune suppressive effects of IDO in the TME. [0493] Figure 25, panels a-g, shows that oxaliplatin-induced ICD provides a successful anti-PDAC vaccination approach. Panel a: Confocal microscopy showing the induction of the ICD marker, CRT, in the presence of PBS, Cis (100 μΜ), OX (50 μΜ), and DOX (1 μΜ) for 4 h in KPC cells. The cell nuclei, surface membrane and CRT were detected by Hoechst 33342, Alexa Fluor® 488-Conjugated Wheat Germ Agglutinin, and Alexa Fluor®647-conjugated anti-CRT antibody staining, respectively. Panel b: CRT surface detection by flow cytometry, using the same conditions and reagents as in (a). Panel c: Animal experimentation using 2 rounds of vaccination one week apart, followed by injecting live KPC cells SC on the contralateral side. The details of the animal vaccination experiment are provided in the methods section. Tumors were harvested on day 29 for IHC and flow cytometry analysis. Panel d: Spaghetti curves to show KPC tumor growth in the contralateral flank. Panel e: Tumor harvesting was performed after animal sacrifice to conduct IHC. Representative images are shown for the IHC staining of CD8 (upper panel) and Foxp3 (lower panel) T cells. The tumor tissues were also analyzed by flow cytometry to determine the CD8/Tregs ratio (see experimental section for details) (right panel). Panel f: IHC staining for cleaved caspase-3 (CC-3) and IFN-γ to demonstrate recruitment of cytotoxic T cells in response to ICD. Panel g: The 3 surviving animals in the OX-treated group, described in panel c, received orthotopic implant of live KPC cells on day 74. Animals maintained their tumor free status up to 132 days, whereupon they were sacrificed for harvesting of immune splenocytes to perform an adoptive transfer experiment. IV injection of the immune splenocytes into the tail vein of B6/129 mice prevented the growth of KPC cells, implanted SC. The controls included IV administration of non-immune splenocytes or saline. The same experiment was also carried out in mice receiving SC injection of B16 melanoma cells. In this case, there was no interference in tumor growth by immune splenocytes in a B6 melanoma model, demonstrating the antigen specificity of the adoptive transfer response (Fig. 33). Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01.
[0494] Figure 26, panels a-f, illustrates synthesis of a self-assembling indoximod
(IND) pro-drug for immune modulatory activity. Panel a: Synthesis route for generating the phospholipid-conjugated IND prodrug (IND-PL). Detailed characterization data are provided in Fig. 34. Panel b: Illustration depicting self-assembly of IND-PL nanovesicles (IND-NV), with IND securely anchored in the lipid bilayer. We also show a representative cryoEM image of the spherical IND-NV, with diameter -80 nm and lipid bilayer thickness of ~7 nm. A lower magnification EM picture is shown in Fig. 34, panel h. Panel c: UPLC -MS/MS to determine the cellular uptake and release of IND-PL. KPC cells were treated with 100 μg/mL free IND or IND-NV for 4, 24, or 72 h, respectively. The data show the fold-increase of the intracellular drug concentration as compared to free IND. A typical UPLC-MS/MS readout is shown in Fig. 35. The detailed sample preparation and analysis are described in Fig. 35. Panel d: Role of IDO-controlled signaling pathways in PDAC immune suppression, and the effect of IND inhibition or perturbation of these pathways (red arrows). mTOR = mammalian target of rapamycin; P-S6K = phosphorylated S6 kinase; AHR = aryl hydrocarbon receptor; Kyn = Kynurenine; GNC2 - 'general control non-derepressible 2". Panel e: KPC cells were treated with free IND or IND-NV at the indicated concentrations for 3 h. Western blot assays showing the enhanced effect of IND-PL on mTOR signaling, leading to the phosphorylation and activation of P-S6 (upper panel). Western blotting also showed enhanced inhibition of AHR expression by the conjugated vs. free drug (lower panel). Panel f: Assessment of IL-6 release into the supernatant by ELISA, demonstrating the enhanced suppressive effect of IND-NV over free drug. Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01.
[0495] Figure 27, panels a-g, shows that co-administration of IND-NV with OX at the tumor site augments anti-PDAC immunity. Panel a: IT co-administration into tumors established by SC injection of KPC cells in syngeneic mice. Treatment details are provided in the methods section. Panel b: KPC tumor growth curve after a single IT injection of the various drugs, when the tumor size approached 60-80 mm3. OX was injected at 1.25 mg/kg. Low (L, 2.5 mg IND /kg) and High (H, 12.5 mg IND /kg) refer to the IND or IND-NV doses. Panel c: Representative tumor images from each group after animal sacrifice on day 31.
Panel d: IHC depicting CD8 and Foxp3 biomarkers in harvested tumor tissue. The full panels of IHC staining data are shown in Fig. 36, panels a-j . Panel e: Flow cytometry determination of CD8/Tregs ratio, as described in Fig. 26, panel e. Panel f: Flow cytometry analysis to determine CD91 expression in the population of CD45+/CD1 lb+/CDl lc+ cells in the tumor tissue. Panel g: IHC to depict CRT and HMGB-1 expression in the harvested tumor tissues. Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01.
[0496] Figure 28, panels a-d, illustrate the development of a dual delivery carrier for OX plus IND using lipid-bilayer coated mesoporous silica nanoparticles (OX/IND-MSNP). Panel a: Schematic to show the structure of OX-laden MSNP, in which the drug is trapped by a lipid bilayer containing IND-PL. This leads to stable entrapment of OX in the pores, with IND-PL trapped in the bilayer. The coating procedure provides uniform and instantaneous sealing of all particle pores. The development of an optimized lipid coating mixture (75% IND-PL, 20% cholesterol and 5% DSPE-PEG2K), is described in Fig. 37, panel a. The CryoEM picture shows a spherical MSNP core and the coated lipid bilayer. Low- magnification cryoEM images are provided in Fig. 37, panel b. Panel b: IVIS optical imaging to study the biodistribution of IV OX/IND-MSNP in orthotopic-implanted KPC tumors in mice (n=6) at the indicated time points. Dylight 680-labeled DMPE was used in the lipid bilayer for NIR imaging. Ex vivo imaging was performed for tumor, heart, liver, spleen, kidney, and lung tissue harvested from the animals 24 and 48 h post injection. Panel c: A separate experiment evaluated the PK profile of OX/IND-MSNP in orthotopic tumor-bearing mice (n=6), receiving single IV injection to deliver the equivalent 5 mg/kg OX and 50 mg/kg IND. Free OX served as a control. Plasma was collected after 0.083, 2, 8, 24 and 48 h, and used for the analysis of IND, IND-PL, and Si content, as described in the methods section in Example 1. Panel d: The tumors and major organs were harvested after 48 for analysis of the tissue content of OX, IND and Si. Results are expressed as mean ± SEM. #p < 0.05.
[0497] Figure 29, panels a-g, shows that dual delivery of OX plus IND-NV by MSNP induced effective anti-PDAC immunity in the orthotopic tumor model. Panel a: Orthotopic tumor-bearing B6/129 mice (n = 7) were IV injected with the OX/IND-MSNP to deliver the equivalent of 5 mg//kg OX and 50 mg/kg IND every 4 days, for a total of 4 administrations. The 1st injection started on day 10. Free OX, OX-MSNP, IND-NV, IND-NV+free OX, and OX/IND-MSNP were used for comparison at the equivalent doses. Interval IVIS imaging monitored tumor growth, which was quantitatively expressed as image intensity at the ROI. Panel b: Representative IVIS imaging on days 10, 18, 27, and 36, according to which the normalized tumor burden was plotted as fold-increase compared to the non-treated control. Panel c: Representative ex vivo bioluminescence imaging on day 36 to show the effect of treatment on metastatic tumor spread to the stomach, intestines, liver, spleen, kidneys, diaphragm, and abdominal wall, but not the heart or lung. We also included in the same experiment, treatment with anti-CD8 and anti-TLR4 antibodies, as well as an injectable siRNA for knockdown of CD91. The effect of interference in the immune response is shown in Fig. 41, panel a. Panel d: Assessment of the survival effect of OX/IND-MS P (n=7) vs. the controls was conducted by repeating the experiment in panel a. Panel e: Serum amylase levels as a reflection of the effect of effective tumor shrinkage by OX/IND-MSNP. Panel f) IHC staining for CD8+ and Foxp3+ T cells in tumor tissue, collected in (c), (left panel).
CD8+/Tregs ratio in tumor tissue determined by flow cytometry (right panel). Panel g: Realtime PCR measurement of P-S6K, AHR, and IL-6 mRNA expression as a result of interference in the IDO pathway. Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01.
[0498] Figure 30, panels a-b, illustrates immuno-PET imaging to demonstrate the induction of the systemic immune response by OX/IND-MSNP administration to animals carrying orthotopic KPC tumors. Panel a: Animals with established orthotopic tumors (n =3/group) were IV inj ected with saline, OX-MSNP (5 mg/kg OX), and OX/IND-MSNP (5 mg/kg OX and 50 mg/ IND on days 10, 14, 18, and 22 post KPC cell implantation into the pancreas. At day 26, 100 μΐ, doses containing 1.07-2.33 MBq (29-63 μϋϊ, 2.3-5.3 μΟ/μ£) 89Zr radiolabeled cDb in saline was IV injected to the same animals. 20 h later, microPET and CT scans were acquired 20 h later by a G8 PET/CT scanner (Sofie Biosciences). Coronal (left panel) and transverse view (right panel) images were acquired and analyzed by AMIDE software. OX/IND-MSNP -treated mice showed significantly increased radioactivity in the tumor, spleen, and TDLN, corresponding to the induction and infiltration of CD8+ T cells. Panel b: To evaluate the CD8+ signal at the tumor site, the operator-defined ROIs were used to demonstrate a 6.2- and 7.5-fold increase in the signal intensity in the tumor interior and periphery, respectively, during OX/IND-MSNP compared to saline treatment. Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01.
[0499] Figure 31, panel a, shows flow cytometry analysis to show the normalized
CRT expression levels in KPC cells exposed to the indicated concentrations of Cis, OX, and DOX for 24 h. Figure 31, panel b, shows the same flow cytometry analysis in PANC-1 cells treated for 4 or 24 h. Figure 31, panel c, shows ELISA measurement of HMGB-1 release from KPC and PANC-1 cells after treatment with Cis, OX, and DOX for 4 h. *p < 0.05; **p < 0.01. [0500] Figure 32, panel a, shows (IVIS optical imaging to follow the tumor burden in the vaccination experiment, as described in Fig. 25, panel d. Figure 32, panel b, shows monitoring of animal weight in the vaccination experiment. Figure 32, panel c, shows IHC analysis to discern CD4 expression in harvested tumor tissue on day 29. [0501] Figure 33 shows the results of an adoptive transfer experiment in which the recipient mice are challenged by SC injection of the B 16 melanoma cell line, after receiving IV injection of immune and non-immune splenocyte populations, as described in Fig. 25, panel g. There was no statistical significance in the growth rate of the 3 different animal groups (n = 6), demonstrating the antigen-specific nature of the anti-PDAC immune response that was not protective against melanoma.
[0502] Figure 34, panels a-i, shows detailed characterization of the intermediary products during IND-PL synthesis, as shown in Fig. 26, panel a. The table in panel a summarizes the intermediates and show the yield at each stage of the synthesis process. Panels, b, and c show ESI-MS, 1H-NMR and 13C-NMR data for N-Boc-IND. Panels d and e show N-Boc-IND-PL analysis by ESI-MS, 1H-NMR and 13C-NMR. Panels f and g show IND-PL analysis by ESI-MS, 1H-NMR and 13C-NMR. Collectively, the comprehensive characterization confirms successful IND-PL synthesis. Panel h shows low magnification cryoEM image of IND-NV. Panel I shows the unfavorable PK of free IND, as demonstrated by the short half-life and low tumor retention capability, and highlights the necessity of a nano-enabled approach to improve retention of the IDO inhibitor.
[0503] Figure 35, panel a, shows UPLC-MS/MS analysis of the cellular content and intracellular release of IND from IND-NV. Figure 35, panel b, shows that the establishment of distinct diluting times for IND (1.23 min), PL (3.97 min), and IND-PL (3.08 min) allowed us to use UPLC analysis to demonstrate the total and released drug content in the cells, as described in Fig. 26, panel c. Figure 35, panel c, shows the results of an abiotic experiment performed to demonstrate that acidic pH and esterase activity can release IND from the prodrug. Briefly, 2 mg/mL of IND-NV was dissolved in 2 mL PBS (pH=7.4), acidified buffer (pH 5.0 and 6), or 100 μΜ esterase in PBS, while vortexing at 37 °C. Solutions were centrifuged and the drug precipitates were dissolved in methanol for overnight before UPLC- MS/MS analysis after 0, 1, 6, 12, 24, 48, 72 h,. *p < 0.05; **p < 0.01; #p < 0.001 compared to pH 7.4 group. [0504] Figure 36, panels a-k, shows the full panel of IHC analysis data in the IT injection experiment (Fig. 27). This includes the staining for: CD8 (panel a), Foxp3 (panel b), CD4 (panel c), CRT (panel d), CD91 (panel e), HMGB-1 (panel f), TLR4 (panel g, left panel), IFN-γ (panel h), IL-10 (panel i), and CC-3 (panel j). We also show H&E staining data in panel (panel K). Moreover, we conducted flow cytometry analysis to quantify TLR4 expression in the population of CD45+ /CD1 lb+ /CD1 lc+ cells (panel g, right panel). *p < 0.05; **p < 0.01. All the immune biomarkers were visualized by DAB (3,3 - Diaminobenzidine), with exception of CD91 that used the Vulcan Fast Red Chromogen Kit 2.
[0505] Figure 37, panel a, illustrates the development and optimization of the IND-PL biofilm to coat MS P with a lipid bilayer. This was accomplished by adjusting the ratio of IND-PL vs. cholesterol and DSPE-PEG2K in the lipid mixture. The optimal molar ratio for IND-PL/Cholesterol/DSPE-PEG2K was determined to be 75:20:5 (ratio #4). Panel b shows nanoparticle characterization of OX/IND-MSNP in DI water, PBS, and DMEM containing 10% FBS on days 1 and 30. OX and IND loading capacities were determined to be 4.4% and 44.3%), respectively. The particles had negligible endotoxin content. We show the lower magnification cryoEM pictures for OX/IND-MSNP.
[0506] Figure 38 shows ICP-OES analysis to quantify the Si content in the orthotopic tumor experiment shown in Fig. 28, panel c. The animals received IV injection of OX/IND- MSNP (5 mg/kg OX; 50 mg/kg IND, 111 mg/kg Si) (n=6). Blood was collected at indicated time points. 48 h post-IV injection, tumor, heart, liver, spleen, kidney, and lung tissues were harvested and weighed. Tissue pieces were digested in HNO3/H2O2 for Si analysis by ICP- OES, as described previously (Liu et al. (2016) ACS Nano, 10(2): 2702-2715). This analysis complements the PK data for the drugs.
[0507] Figure 39, panel a, shows representative autopsy images of the animals treated with the dual delivery MSNP, as described in Fig. 29, panel c. The broken lines highlight the primary tumors and their local invasion, while the arrows point to metastases. Figure 39, panel b, shows a heat map summary of the ex vivo imaging data after tumor and organ harvesting for the experiment shown in Fig. 29, panel c. This demonstrates a significant reduction in tumor metastases in response to OX/IND-MSNP treatment, and to a lesser extent OX-MSNP.
[0508] Figure 40, panels a-k, shows the full panel of IHC data collected during the systemic delivery experiment in Fig. 29, showing results for the following markers: CD8 (panel a), Foxp3 (panel b), CRT (panel c), CD91 (panel d, left panel), HMGB-1 (e), TLR4 (panel f, left panel), IFN-γ (panel g), perforin (panel h), IL-10 (panel i), and CC-3 (panel j). We also included flow cytometry analysis of CD91 positive cells in the CD45+ /CD1 lb+ /CD1 lc+ cell population in the lower panel in panel d, and the same analysis for TLR4 and CD 103 positive cells in similarly phenotyped cells in the lower panel in panels f, and k, respectively. *p < 0.05; **p < 0.01.
[0509] Figure 41, panels a-d, shows a demonstration of the impact of IV injection of antibodies to CD8 and TLR-4, or an injectable pool of siRNAs targeting CD91 in the systemic biodistribution experiment described in Fig. 29. Panel a: Representative IVIS tumor imaging on days 10, 18, 27, and 36 from mice receiving OX/IND-MSNP (5 mg/kg OX and 50 mg/kg IND) with or without treatment, anti-CD8, anti-TLR4 or CD91 siRNA knockdown (n =7). The normalized tumor burden, as reflected by the luminescence intensity in the ROI, was plotted and displayed in the right side panel. Panel b: Representative autopsy results and ex vivo bioluminescence imaging intensity is depicted to show the impact of interference in the immune response on tumor growth and metastases. Panel c: Animal survival rate in the same experiment. Panel d: Impact on the CD8/Treg ratio in the same experiment.
Collectively, these data show that interference in both innate (CD91, TLR40) and adaptive (CD8) immunity could significantly reverse the antitumor efficacy of OX/IND-MSNP. *p < 0.05; **p < 0.01. [0510] Figure 42 panel A, shows monitoring of animal weight during treatment with the dual delivery carrier in Fig. 29. Fig. 42, panel B, shows monitoring of liver enzymes (ALT, AST, and ALP) in the orthotopic tumor model IV described in Figs. 29 and 41.
OX/IND-MSNP dual delivery particles did not exert toxicity during the experiment. Instead, it helped to protect against liver toxicity from IV injected free OX. Similarly, there was no biochemical evidence of toxicity in the kidney or heart (data not shown). **p < 0.01.
[0511] Figure 43 shows normalized area of interest (ROI) scanning data to show the radio label portioning to the spleen and tumor draining lymph nodes (TDLN)in mice treated by saline, OX-MSNP, and OX/IND-MSN,P followed by IV immuno-PET particle (89Zr- malDFO-169 cDb) injection, as described in Fig. 30. *p < 0.05; **p < 0.01. [0512] Figures 44A-44C illustrate the screening of nanomaterial (NM)-induced immunogenic cell death (ICD) in KPC pancreatic cancer cell after 24 h treatment with engineered nanoparticles. Figure 44A illustrates the induction of calreticulin (CRT) following treatment with a nanomaterial {see, e.g., Table 10 in Example 7). Figure 44B shows dose and time-dependent CRT induction in KPC cells. Figure 44C shows the high mobility group box 1 protein (FDVIGB-1) concentration in the supernatant of the KPC cells after being treated with various NMs. [0513] Figures 45 shows cytotoxicity profile of metal oxides (MOs), graphene oxides
(GO), and carbon nanotubes (CNT) in KPC cells after 24 h treatment.
[0514] Figures 46A and 46B, show the results of a vaccination experiment using metal and metal oxide. Fig. 46A) Animal experimentation using 2 rounds of vaccination (dying KPC cells treated with metal oxide nanoparticles) one week apart, followed by injecting live KPC cells SC on the contralateral side. Spaghetti curves to show KPC tumor growth in the contralateral flank. Fig. 46B: IVIS imaging to monitor the tumor growth on the contralateral flank of mice shown in Fig. 46A. At the conclusion stage, the tumor tissues were used for flow cytometry experiment to measure CD8/Treg cell ratios.
[0515] Figures 47A-47C show the results of an intratumoral injection (IT) experiment using metal and metal oxide nanoparticles. KPC cells were subcutaneously injected into B6/129 mice. Fig. 47A) Dose-seeking experiment for CuO nanoparticle. The subQ tumors received single IT injection of CuO nanoparticle at 15, 30, 50 and 100 ug/mouse. The tumors were monitored up to 23 days. We used 50 ug CuO/mouse in the following IT experiment. Fig. 47B) In a pilot efficacy study using IT injection, KPC subQ tumor mice received single IT injection using indicated NMs. The doses were shown in the figure. Tumor growth was monitored up to -23 days. Fig. 47C). On day 23, the tumors were harvested and single cell suspension was collected for flow analysis of various immune biomarkers. Significantly enhanced antitumor immunity was found in CuO group as confirmed by the boosted
CD8/Treg ratio, granzyme B, IFN-gamma, etc. The IHC staining of tumor tissue was ongoing.
[0516] Figures 48, panels A-E, shows the results of an intratumoral injection (IT) experiment using GOs. Panel A: Schematically illustrates the vaccination protocol (similar vaccination study in Fig. 46). KPC cells were treated with GOs. The dying cells were used to vaccinate the mice. Panel B: Tumor volume as a function of time pose live KPC for implantation. Panel C: IVIS imaging to monitor the KPC tumor growth on the contralateral flank of the mice. Panel D: The CD8/Treg cell ratio determined by flow cytometry. Panel E) Selective IHC staining in the GO vaccination experiment, such as CD8, Fox-P3, CRT, Caspase 3 (CC3) and Perforin, are shown.
[0517] Figures 49A-49C show the results of an intratumoral injection (IT) experiment using GOs. Figure 49A: KPC subQ tumor mice received single IT injection using indicated GOs. The doses were shown in the figure. Tumor growth was monitored up to -23 days. Figure 49B: At the conclusion stage, the tumor samples were harvested for CD8/Treg ratio measurement by flow cytometry. Figure 49C: IP of anti-CD8 mAb (200 ug/mouse) interferes the ICD-mediated tumor inhibition induced by small GO.
[0518] Figure 50 illustrates mass spectrometry of 1-L-MT-PL. [0519] Figure 51 illustrates the intracellular uptake of 1 -L-MT-PL in KPC cells at varied time points. UPLC-MS/MS was performed to determine the cellular uptake and release of 1-L-MT-PL. KPC cells were treated with 100 μg/mL nanovesicles, formed by 1-L- MT-PL. Controls include free 1-L-MT, free 1-D-MT and nanovesicles made by 1-D-MT-PL. After indicated incubation period, the cells were collected via trypsinization and drug extraction, as described by Lu et al. (2017) Nat. Comm. 8: 1811). The data show the fold- increase of the intracellular drug concentration as compared to free 1-L-MT.
[0520] Figure 52 illustrates western blot and ELISA for P-S6K (cell lysate) and IL-6
(supernatant) in KPC cells treated with 1-L-MT-PL at 10 μΜ and 50 μΜ in tryptophan- deficient medium. KPC cells were treated with 1-L-MT-PL nanovesicles at the indicated concentrations for 3 h in tryptophan-deficient DMEM. Controls include free 1-L-MT, free 1- D-MT and nanovesicles made by 1-D-MT-PL. Western blot assays showing the enhanced effect of 1-L-MT-PL on mTOR signaling, which can be studied by assessing the
phosphorylation of P-S6K (upper left panel). The graphic in the lower right panel shows the pooled data for 3 experiments to assess P-S6K activation at 10 μΜ and 50 μΜ 1-MT. The supernatants from the 3 experiments shown in P-S6K western assays were collected for IL-6 measurement using ELISA. This allows us to demonstrate the inhibition of the
IL6/STAT3/AHR autocrine signaling loop using 1-L-MT-PL, similar to 1-D-MT-PL.
[0521] Figure 53 illustrates the effect of different isomers in an in vitro IDO enzymatic assay in 4T1 breast cancer cells. The experiment procedure is similar to literature (Hou et al. (2007) Cancer Res. 67(2): 792-801). lxlO5 4T1 cells were seeded into 24-well plate. After cell attachment, the cells were treated with different 1-MT isomers or prodrugs at various concentrations. Meanwhile, 100 ng/ml of mouse recombinant IFN-γ was added per well to stimulate IDO expression. Then, the plates were incubated for 20 hr at 37°C in a humidified C02 incubator. Supernatants were harvested and analyzed for kynurenine by ELISA. The anti-cancer efficacy experiments were performed in vivo.
DETAILED DESCRIPTION
[0522] In various embodiments three treatment modalities are provided to generate an anti-cancer response premised, inter alia, on the induction of immunogenic cell death (ICD) in cancer cells. ICD is responsible for enhanced tumor antigen presentation as well as providing stimulatory effects to the participating DCs. This can trigger the activation of cytotoxic T cells and anti-cancer {e.g., anti-PDAC) immunity that can be synergistically enhanced by an intervention in the IDO pathway.
[0523] A first treatment modality involves combination of an ICD inducer {e.g., oxaliplatin) in combination with an IDO inhibitor {e.g., indoximod) into a single nanocarrier that allows systemic (or local) biodistribution and drug delivery to tumor sites. The dual- delivery approach achieved synergistic enhancement of adaptive and innate immunity {e.g., anti-PDAC immunity), leading to a significant improvement in animal survival. In certain embodiments the nanocarrier comprises a vesicle {i.e., a lipid bilayer enclosing a fluid). In certain embodiments the nanocarrier comprises a nanoparticle {e.g., a mesoporous silica nanoparticle (MSNP) surrounded (encapusulated) by a lipid bilayer.
[0524] A second treatment modality involves local delivery to a tumor or peri- tumoral region, of an agent that induces ICD {e.g., oxaliplatin) in combination with a lipid
{e.g., a nanovesicle) that comprises an inhibitor of the IDO pathway {e.g., indoximod). It was demonstrated that such local delivery of an ICD inducer in combination with an IDO inhibitor induces recruitment of cytotoxic CD8+ lymphocytes, depletion of Tregs, reversal of the CD8+/Foxp3+ ratio, cytotoxic tumor killing, and tumor shrinkage at the local site. These adaptive immune responses were accompanied by boosting of the innate immune system, as reflected by CRT and HMGB1 expression, as well as the activation of a DC population, particularly well-suited for generating cytotoxic T cell responses.
[0525] A third treatment modality involves vaccination utilizing dying cancer cells
{e.g., KPC cells) in which ICD is induced ex vivo. It was discovered that such vaccination can generate a systemic immune response that can interfere with tumor growth at a remote site as well as allowing adoptive transfer to non-immune animals. [0526] In various embodiments, methods and compositions for performing these treatment modalities are provided.
Approach 1— Systemic treatment of a cancer by combined delivery of ICD and IDO inhibition.
[0527] The first approach approach combines an ICD-inducer (e.g., doxirubicin, oxaliplatin, etc.) and an inhibitor of the IDO pathway (e.g., indoximod) into a single nanocarrier, that can provide systemic biodistribution and drug delivery to orthotopic tumor sites.
[0528] In certain embodiments this dual-delivery approach involves the formation of lipid vesicles where a component of the lipid bilayer comprising the vesicle incorporates or is conjugated to an inhibitor of the IDO pathway (e.g., an indoximod prodrug such as IND-PL) and the vesicle contains an ICD inducer (e.g., doxorubicin (DOX)). This approach is illustrated herein in Examples 2 and 3.
[0529] In another illustrative, but non-limiting embodiment, the nanocarrier comprises a mesoporous silica nanoparticle (MS P) containing the ICD inducer (e.g., oxaliplatin) where the silica nanoparticle is surrounded by (encapsulated by ) a lipid bilayer containing (or conjugated to) an IDO inhibitor (e.g., indoximod provided as the prodrug IND- PL (Formula I)). The lipid bilayer (LB) coated MSNP, also known as a silicasome (see, e.g., PCT Patent Application No: PCT/US2017/012625) provides effective dual delivery of the ICD inducer and IDO inhibitor. Thus, as shown in Example 5, this dual-delivery approach achieved synergistic enhancement of adaptive and innate anti-PDAC immunity, leading to a significant improvement in animal survival.
[0530] A third dual-delivery approach exploits the discovery that certain
nanomaterials (e.g., CuO, Cu20, Sb203, As203, Bi203, P203, ZnO, Ti02, graphene oxide, 2D materials other than graphene or graphene oxide (e.g., graphene, graphyne, borophene, germanene, silicene, Si2BN, stanene, phosphorene, bismuthene, molybdenite, metals, 2D supracrystals, and the like), can induce immunogenic cell death (ICD) (see, e.g., Example 7). Nanoparticles formed from these ICD inducers can readily be coated with a lipid that contains (or is conjugated to) an IDO inhibitor (e.g., indoximod provided as the prodrug IND- PL (Formula I)). The lipid coated nanomaterial thus forms a dual delivery vehicle for delivery of both an ICD-inducer and an IDO-inhibitor. Accordingly, in certain embodiments, the following dual-delivery vehicles are contemplated herein: [0531] 1) ICD-inducer/IDO-inhibitor vesicle;
[0532] 2) ICD-inducer/IDO-inhibitor silicasome (LB-coated nanoparticle);
[0533] 3) ICD-inducer nanomaterial (bioreactive nanomaterial) coated with IDO- inhibitor lipid (phospholipid prodrug). [0534] It will be recognized, that in addition to systemic administration, any of these carriers are suitable for local treatment of a tumor. Thus, for example, any of these carriers can be administered topically (e.g., for skin tumors), or directly, e.g., to an intra-tumoral or peri -tumoral site, e.g., via injection or during a surgical procedure.
Dual-Delivery Lipid Vesicles (e.g. , ICD IDO inhibitorVesicles)
[0535] In certain embodiments dual-delviery nanovesicles are provided for the delivery of an ICD-inducer in combination with an inhibitor of the IDO pathway and/or for the delivery of an ICD inducer and a pharmacological agent other than an ICD inducer or in combination with an ICD inducer in addition to the inhibitor of the IDO pathway.
[0536] Accordingly, in certain embodiments, a nanovesicle drug carrier for the combined delivery of an inhibitor of an IDO pathway and an inducer of immunogenic cell death (ICD), is provided where the nanovesicle drug carrier comprises a lipid vesicle where a lipid bilayer effectively forms a vesicle in an aqueous solution, and the lipid or lipid formuation comprising the vesicle is associated with (or conjugated to) an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway (IDO pathway inhibitor); and a cargo within the vesicle where the cargo comprises an agent that induces immunogenic cell death (ICD) (ICD- inducer). It is noted that while this embodiment is described with respect to a cargo that induces immunogenic cell death, other cargos are contemplated as an alternative or in addition to the ICD inducer. Such cargos include, inter alia, various cancer
chemotherapeutics as described herein. The lipid vesicle is typically formed from a lipid bilayer. However in certain embodiments, a lipid micelle (which does not comprises a lipid bilayer) is contemplated. Thus, for example, in certain embodiments a lipid micelle can be comprise a phospholipid prodrug {e.g., lipid-IDO pathway inhibitor conjugate) and a cargo (typically a lipophilic) cargo can be disposed inside the micelle. In certain embodiments the nanovesicle provides an IDO inhibitor and an ICD inducer that are synergistic in their activity against a cancer. In certain embodiments the nanovesicle drug carrier, when administered systemically, delivers an amount of an ICD inducer effective to induce or to facilitate induction of immunogenic cell death of cancer cells at the tumor site. In certain embodiments the nanovesicle drug carrier, when administered systemically, delivers an amount of IDO inhibitor to partially or fully inhibit an IDO pathway at a cancer site.
[0537] In certain embodiments the inhibitor of the IDO pathway comprises an agent selected from the group consisting of 1-methyl-D-tiyptophan (indoximod, D-1MT), L-1MT, methylthiohydantoin-dl -tryptophan (MTH-Trp, Necrostatin), β-carbolines (e.g., 3-butyl-P- carboline), naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassinin, N- [2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S- methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl- brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol-3-yl)ethyl]- S[(naphth-2-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-3-yl)methyl]- dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-4-yl)methyl]-dithiocarbamate, 5-bromo- brassinin, Phenylimidazole-based IDO inhibitors (e.g., 4-phenylimidazole), Exiguamine A, imidodicarbonimidic diamide,N-methyl-N'-9-phenanthrenyl-monohydrochloride
(NSC401366), INCB024360 (Epacadostat), l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDO 1 -derived peptide, LG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole. In certain embodiments the IDO inhibitor comprises indoximod. In certain embodiments the IDO inhibitor comprises substantially pure "L" indoximod or substantially pure "R" indoximod, or a racemic mixture of "D" and "L" indoximod. [0538] In certain embodiments the inhibitor of the IDO pathway, is disposed in a lipid comprising the vesicle and/or conjugated to a lipid comprising said vesicle. In certain embodiments the vesicle comprises a phospholipid. In certain embodiments the vesicle comprises a phospholipid, and cholesterol (CHOL). In certain embodiments the phospholipid comprises a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C 12-C20 carbon chains. In certain embodiments the phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC),
dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), and diactylphosphatidylcholine (DAPC). In certain embodiments the phospholipid comprises a natural lipid selected from the group consisting of egg phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC). In certain embodiments the phospholipid comprises an unsaturated fatty acid selected from the group consisting of l,2-dimyristoleoyl-sn-glycero-3- phosphocholine, 1 ,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine, 1 ,2-dioleoyl-sn-glycero-3 - phosphocholine (DOPC), and l,2-dieicosenoyl-sn-glycero-3-phosphocholine. In certain embodiments the vesicle comprises an mPEG phospholipid with a phospholipid C14-C18 carbon chain, and a PEG molecular weight ranging from about 350 Da to 5000 Da. In certain embodiments the vesicle comprises l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (DSPE-PEG). In certain embodiments the vesicle comprises DPSE-PEG2K- In certain embodiments the IDO inhibitor is conjugated to a component of said vesicle. In certain embodiments the IDO inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid. In certain embodiments the IDO inhibitor is conjugated directly to the moiety, while in other embodimetns, the IDO inhibitor is conjugated to the moiety via a linker. In certain embodiments the IDO inhibitor is conjugated to PGHP. In certain embodiments the IDO inhibitor is conjugated to vitamin E. In certain embodiments the IDO inhibitor is conjugated to cholesterol (CHOL), or squalene. In certain embodiments the IDO inhibitor is conjugated to a fatty acid (e.g., oleic acid or docosahexaenoic acid). In certain embodiments the inhibitor of the IDO pathway is conjugated to oleic acid or docosahexaenoic acid via an HO-(CH2)n=2-5-OH linker. In certain embodiments the inhibitor of the IDO pathway is conjugated to a lipid. In certain
embodiments the inhibitor of the IDO pathway is conjugated to a phospholipid comprising the lipid vesicle. In certain embodiments the inhibitor of the IDO pathway is conjugated to 1- palmitoy l -2-hydroxy-sn-glycero-3-phosphocholine (PL) (e.g., IND-PL, Formula I). [0539] In certain embodiments the bilayered vesicle comprises IND-PL/Chol/DSPE-
PEG. In certain embodiments the vesicle comprises about 75% IND-PL, about 20% cholesterol, and about 5% DSPE-PEG2K. In certain embodiments the ICD inducer comprises a chemotherapeutic agent selected from the group consisting of doxorubicin, oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide. In certain
embodiments the ICD inducer comprises doxorubicin.
Dual-Delivery (ICD-inducer/IDO-inhibitor) LB Coated MSNPs (ICD/IDO Silicasomes).
[0540] As noted above, in certain embodiments a dual delivery carrier for an ICD inducer (e.g., oxaliplatin) and an IDO inhibitor (e.g., indoximod) was developed where the carrier comprises lipid-bilayer coated nanoparticles (e.g., mesoporous silica nanoparticles). In various illustrative embodiments, the IDO inhibitor (e.g., indoximod) is provided disposed in and/or conjugated to a component of the lipid bilayer while the ICD inducer is provided on or in (e.g., within the pores) of the nanoparticle, e.g., effectively sealed/contained within the lipid bilayer. However, it will be recognized that in certain embodiments the ICD inducer can be provided in or conjugated to the lipid bilayer while the IDO inhibitor is contained on or within the nanoparticle. Such lipid bilayer coated nanoparticle drug delivery systems (aka silicasomes), are capable of delivering two (or more) active agents in precise concentration ratios as desired.
[0541] In one illustrative, but non-limiting embodiment the "dual-delivery carrier" comprises indoximod conjugated to a component of the lipid bilayer (e.g., as IND-PL (Formula I)) while the ICD inducer (e.g., oxaliplatin) is disposed within the nanoparticle. This leads to stable entrapment of the ICD-inducer (e.g., OX) in the pores, with IND-PL trapped in the bilayer. The coating, procedure(s) described herein provide uniform and instantaneous sealing of all particle pores (see, e.g., Examples 5 and 2).
[0542] Accordingly in certain embodiments, a nanoparticle drug carrier for the combined delivery of an inhibitor of an IDO pathway and an inducer of immunogenic cell death (ICD) is provided where the nanoparticle drug carrier comprises: a mesoporous silica nanoparticle having a surface and defining a plurality of pores that are suitable to receive molecules therein; a lipid bilayer coating the surface; a first cargo comprising an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway (IDO inhibitor); and a second cargo comprising an agent that induces immunogenic cell death (ICD) (ICD-inducer); where the lipid bilayer is substantially continuous and encapsulates the nanoparticle stably sealing the plurality of pores. In certain embodiments the nanoparticle drug carrier contains a predefined ratio of IDO inhibitor to ICD-inducer. As illustrated herein in the Examples, in certain embodiments, the IDO inhibitor and the ICD inducer are synergistic in their activity against a cancer (e.g., against PDAC). [0543] In various embodiments the drug carrier, when administered systemically, is effective to deliver an amount of an ICD inducer effective to initiate or to facilitate induction of immunogenic cell death of a cancer cell. In certain embodiments the drug carrier, when administered systemically, is effective to deliver an amount of IDO inhibitor to partially or fully inhibit an IDO pathway at a cancer site. In certain embodiments, where the activity of the ICD inducer and IDO inhibitor is synergistic, the drug carrier can contain/provide a lower dose ICD inducer and/or IDO inhibitor than when these agents are used individually. In certain embodiments the combination of the ICD inducer and the IDO inhibitor achieves an anti-cancer activity that cannot be achieved by the use of either agent alone. [0544] In certain embodiments the IDO inhibitor is disposed in the lipid bilayer and/or conjugated to a lipid comprising said lipid bilayer while the ICD inducer is disposed in the plurality of pores. In certain embodiments the ICD-inducer comprises a chemical or biological agent described in Table 2, above. In certain embodiments the ICD-inducer comprises a chemotherapeutic agent selected from the group consisting of oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide. In certain embodiments the ICD-inducer comprises oxaliplatin.
[0545] In certain embodiments the ICD inducer comprises an ICD inducing nanomaterial (e.g., CuO, Cu20, Sb203, As203, Bi203, P203, ZnO, Ti02, graphene oxide, 2D materials other than graphene or graphene oxide (e.g., graphene, graphyne, borophene, germanene, silicene, Si2BN, stanene, phosphorene, bismuthene, molybdenite, metals, 2D supracrystals, and the like) as described above or in Example 7. In certain embodiments, the ICD-inducing nanomaterial can be contained on or within the nanoparticle. In certain embodiments an ICD-inducing nanomaterial can be coated with a lipid or with a lipid bilayer. In certain embodiments the ICD-inducing nanomaterial can incorporate one or more drugs as described herein. In certain embodiments, where the ICD-inducing nanomaterial is within a lipid bilayer the nanomaterial may contain the IDO inhibitor, both of which can be released at a target site (e.g., cancer cell). In certain embodiments, where the ICD-inducing
nanomaterial comprises graphene oxide, the surface can be functionalized to deliver the IDO- inhibitor.
[0546] In certain embodiments, the IDO inhibitor comprises an agent selected from the group consisting of 1-methyl-D-tiyptophan (indoximod, D-1MT), L-1MT,
methylthiohydantoin-dl -tryptophan (MTH-Trp, Necrostatin), β-carbolines (e.g., 3-butyl-P- carboline), Naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S- methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl- brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol-3-yl)ethyl]- S[(naphth-2-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-3-yl)methyl]- dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-4-yl)methyl]-dithiocarbamate, 5-bromo- brassinin, Phenylimidazole-based IDO inhibitors (e.g., 4-phenylimidazole), Exiguamine A, imidodicarbonimidic diamide,N-methyl-N'-9-phenanthrenyl-monohydrochloride
(NSC401366), INCB024360 (Epacadostat), l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDO l -derived peptide, LG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole. In certain embodiments the IDO inhibitor comprises an agent shown in Table 3, above. In certain embodiments the IDO inhibitor comprises indoximod. [0547] In certain embodiments, the nanoparticle drug carrier is fabricated so that a population of the drug carriers in suspension shows essentially a substantially unimodal size distribution; and/or shows a PDI less than about 0.2, or less than about 0.1 ; and/or shows a coefficient of variation in size less than about 0.1 or less than about 0.05. In certain embodiments, the nanoparticle drug carriers distribute to developing tumor sites on IV injection. In certain embodiments the nanoparticle drug carrier forms a stable suspension on rehydration after lyophilization. In certain embodiments the nanoparticle drug carriers, show reduced drug toxicity as compared to free drug and/or drug in liposomes. In certain embodiments the nanoparticle drug carrier has colloidal stability in physiological fluids with pH 7.4 and remains monodisperse to allow systemic biodistribution and is capable of entering a disease site by vascular leakage (EPR effect) or transcytosis.
[0548] Various nanoparticle (e.g., mesoporous silica core), lipid bilayer formulations, and methods of synthesis are described in the sections below and in the examples.
Nanoparticles.
[0549] In various embodiments silicasome drug carriers described herein comprise a porous silica (or other material) nanoparticle (e.g., a silica body having a surface and defining a plurality of pores that are suitable to receive molecules therein) coated with a lipid bilayer. For example, in certain embodiments the silica nanoparticle can be a mesoporous silica nanoparticle. The fact that the nanoparticle is referred to as a silica nanoparticle does not preclude materials other than silica from also being incorporated within the silica
nanoparticle. In some embodiments, the silica nanoparticle may be substantially spherical with a plurality of pore openings through the surface providing access to the pores. However, in various embodiments the silica nanoparticle can have shapes other than substantially spherical shapes. Thus, for example, in certain embodiments the silica nanoparticle can be substantially ovoid, rod-shaped, a substantially regular polygon, an irregular polygon, and the like.
[0550] Generally, the silica nanoparticle comprises a silica body that defines an outer surface between the pore openings, as well as side walls within the pores. The pores can extend through the silica body to another pore opening, or a pore can extend only partially through the silica body such that that it has a bottom surface of defined by the silica body.
[0551] In some embodiments, the silica body is mesoporous. In other embodiments, the silica body is microporous. As used herein, "mesoporous" means having pores with a diameter between about 2 nm and about 50 nm, while "microporous" means having pores with a diameter smaller than about 2 nm. In general, the pores may be of any size, but in typical embodiments are large enough to contain one or more therapeutic compounds therein. In such embodiments, the pores allow small molecules, for example, therapeutic compounds such as anticancer compounds to adhere or bind to the inside surface of the pores, and to be released from the silica body when used for therapeutic purposes. In some embodiments, the pores are substantially cylindrical.
[0552] In certain embodiments the nanoparticles comprise pores having pore diameters between about 1 nm and about 10 nm in diameter or between about 2 nm and about 8 nm. In certain embodiments the nanoparticles comprise pores having pore diameters between about 1 nm and about 6 nm, or between about 2 nm and about 5 nm. Other embodiments include particles having pore diameters less than 2.5 nm. In other
embodiments, the pore diameters are between 1.5 and 2.5 nm. Silica nanoparticles having other pore sizes may be prepared, for example, by using different surfactants or swelling agents during the preparation of the silica nanoparticles. [0553] In various embodiments the nanoparticles can include particles as large (e.g., average or median diameter (or other characteristic dimension) as about 1000 nm. However, in various embodiments the nanoparticles are typically less than 500 nm or less than about 300 nm as, in general, particles larger than 300 nm may be less effective in entering living cells or blood vessel fenestrations. In certain embodiments the nanoparticles range in size from about 40 nm, or from about 50 nm, or from about 60 nm up to about 100 nm, or up to about 90 nm, or up to about 80 nm, or up to about 70 nm. In certain embodiments the nanoparticles range in size from about 60 nm to about 70 nm. Some embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 1000 nm. Other embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 500 nm. Other embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 200 nm. In some
embodiments, the average maximum dimension is greater than about 20nm, greater than about 30nm, greater than 40nm, or greater than about 50nm. Other embodiments include nanoparticles having an average maximum dimension less than about 500 nm, less than about 300nm, less than about 200nm, less than about 100 nm or less than about 75 nm. As used herein, the size of the nanoparticle refers to the average or median size of the primary particles, as measured by transmission electron microscopy (TEM) or similar visualization technique.
[0554] Illustrative mesoporous silica nanoparticles include, but are not limited to
MCM-41, MCM-48, and SB A- 15 (see, e.g., Katiyaret a/. (2006) J. Chromatog. 1122(1-2): 13-20).
[0555] Methods of making porous silica nanoparticles are well known to those of skill in the art. In certain embodiments mesoporous silica nanoparticle are synthesized by reacting tetraethyl orthosilicate (TEOS) with a template made of micellar rods. The result is a collection of nano-sized spheres or rods that are filled with a regular arrangement of pores. The template can then be removed by washing with a solvent adjusted to the proper pH (see, e.g., Trewyn et al. (2007) Chem. Eng. J. 137(1): 23-29. In certain embodiments mesoporous particles can also be synthesized using a simple sol-gel method (see, e.g., Nandiyanto, et al. (2009) Microporous and Mesoporous Mat. 120(3): 447-453, and the like). In certain embodiments tetraethyl orthosilicate can also be used with an additional polymer monomer (as a template). In certain embodiments 3-mercaptopropyl)trimethoxysilane (MPTMS) is used instead of TEOS. [0556] In certain embodiments the mesoporous silica nanoparticles are cores are synthesized by a modification of the sol/gel procedure described by Meng et al. (2015) ACS Nemo, 9(4): 3540-3557. To synthesize a batch of -500 mg of MS P, 50 mL of CTAC is mixed with 150 mL of H20 in a flask (e.g., a 500 mL conical flask), followed by stirring (e.g., at 350 rpm for 15 min at 85°C). This us followed by the addition of 8 mL of 10% triethanolamine for 30 min at the same temperature. Then, 7.5 mL of the silica precursor, TEOS, is added dropwise at a rate of 1 mL/min using a peristaltic pump. The solution is stirred at 350 rpm at 85°C for 20 min, leading to the formation particles with a primary size of ~65 nm. The surfactant can be removed by washing the particles with a mixture of methanol/HCl (500: 19 v/v) at room temperature for 24 h. The particles can be centrifuged at 10 000 rpm for 60 min and washed three times in methanol.
[0557] While the methods described herein have been demonstrated with respect to porous silica nanoparticles (e.g., mesoporous silica), it will be recognized that similar methods can be used with other porous nanoparticles. Numerous other mesoporous materials that can be used in drug delivery nanoparticles are known to those of skill in the art. For example, in certain embodiments mesoporous carbon nanoparticles could be utilized.
Mesoporous carbon nanoparticles are well known to those of skill in the art (see, e.g., Huang et al. (2016) Carbon, 101 : 135-142; Zhu et al. (2014) Asian J. Pharm. Sci., 9(2): 82-91; and the like).
[0558] Similarly, in certain embodiments, mesoporous polymeric particles can be utilized. The syntheses of highly ordered mesoporous polymers and carbon frameworks from organic-organic assembly of triblock copolymers with soluble, low-molecular-weight phenolic resin precursors (resols) by an evaporation induced self-assembly strategy have been reported by Meng et al. (2006) Chem. Mat. 6(18): 4447-4464 and in the references cited therein.
[0559] The nanoparticles described herein are illustrative and non-limiting. Using the teachings provided herein numerous other lipid bilayer coated nanoparticles will be available to one of skill in the art.
Lipid Bilayer and Methods of Coating Nanoparticles With a Lipid Bilayer.
[0560] The drug carrier nanoparticles described herein comprise a porous
nanoparticle (e.g. a mesoporous silica nanoparticle (MSNP)) coated with a lipid bilayer. In certain embodiments the bilayer composition is optimized to provide a rapid and uniform particle coating, to provide colloidal and circulatory stability, and to provide effective cargo retention, while also permitting a desirable cargo release profile.
[0561] In certain embodiments the lipid bilayer comprises a combination of a phospholipid, cholesterol, and in certain embodiments, a IDO-lipid conjugate, a pegylated lipid (e.g., DSPE-PEG2000), or a factionalized pegylated lipid (e.g., DSPE-PEG2000- maleimide) to facilitate conjugation with targeting or other moieties.
[0562] To attach a surface LB coating, a coated lipid film procedure can be utilized in which MSNP suspensions are added to a large lipid film surface, coated on, e.g., a round- bottom flask. Using different lipid bilayer compositions, a series of experiments can be performed to find a composition and optimal lipid/particle ratio that provides rapid and uniform particle wrapping, coating and effective cargo retention and/or release upon sonication. It is believed that this lipid composition and wrapping cannot be achieved by liposomal fusion to the particle surface under low energy vortexing conditions.
[0563] As described in Example 5, in certain embodiments, the mesoporous silica nanoparticles are coated with a lipid bilayer that incorporates the IDO inhibitor coupled to a lipid (e.g., a phospholipid) or to cholesterol. In one illustrated embodiment the mesoporous silica nanoparitcles are coated with a lipid bilayer comprising IND-PL, as well as serving to encapsulate oxaliplatin in the porous interior (Figure 28, panel a).
[0564] Careful experimentation was undertaken to establish the optimal lipid bilayer composition for an OX/IND drug delivery carrier (e.g., a bilayer coated nanoparticle). This was accomplished by using an IND-PL/Cholesterol/DSPE-PEG2K mixture at a molar ratio of 75:20:5 (Figure 37, panel a). The biofilm was laid down at the bottom of a round bottom flask, to which the OX-soaked MSNPs were added, followed by sonication (see, e.g., Liu et al. (2016) ACS Nano, 10(2): 2702-2715; Meng et al. (2015) ACS Nano, 9(4): 3540-3557).
[0565] The lipid bilayer formulation described above and in Example 5 is illustrative and non-limiting. Depending on the drug(s) being loaded into the drug delivery carrierand the desired release profile, in various embodiments different lipid bilayer formulations can be used and an optimal formulation can be determined. Thus, while the described molar ratio of 75:20:5 is optimized for the particular combination of oxaliplatin and indoximod, using the methods described in Example 5, the lipid bilayer can routinely be optimized for other combinations of ICD inducer and IDO inhibitor, and lipid bilayer components.
[0566] Accordingly, in certain embodiments the lipid bilayer can comprise: 1) one or more saturated fatty acids with C14-C20 carbon chain, such as
dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and diactylphosphatidylcholine (DAPC); and/or 2) One or more unsaturated fatty acids with a C14-C20 carbon chain, such as 1,2- dimyristoleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine,l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dieicosenoyl-sn- glycero-3-phosphocholine; and/or 3) Natural lipids comprising a mixture of fatty acids with C12-C20 carbon chain, such as Egg PC, and Soy PC, sphingomyelin, and the like. These lipids are illustrative but non-limiting and numerous other lipids are known and can be incorporated into a lipid bilayer for formation of a drug delivery nanocarrier (e.g., a bilayer- coated nanoparticle). [0567] In certain embodiments the drug carrier comprises bilayer comprising a lipid
(e.g., a phospholipid), cholesterol, and a PEG functionalized lipid (e.g., a mPEG
phospholipid). In certain embodiments the mPEG phospholipids comprises a C14-C18 phospholipid carbon chain from, and a PEG molecular weight from 350-5000 (e.g., MPEG 5000, MPEG 3000, MPEG 2000, MPEG 1000, MPEG 750, MPEG 550, MPEG 350, and the like). In certain embodiments the mPEG phospholipid comprises DSPE-PEG5000, DSPE- PEG3000, DSPE-PEG2000, DSPE-PEG1000, DSPE-PEG750, DSPE-PEG550, or DSPE- PEG350. MPEGs are commercially available (see, e.g., //avantilipids.com/product- category/products/polymers-polymerizable-lipids/mpeg-phospholipids). [0568] In certain embodiments lipid bilayer comprises an mPEG phospholipid with a phospholipid C14-C18 carbon chain, and a PEG molecular weight ranging from about 350 Da to 5000 Da. In certain embodiments the lipid bilayer comprises DPSE-PEG2K.
[0569] In certain embodiments the lipid bilayer comprises 1,2-distearoyl-sn-glycero-
3 -phosphoethanolamine-PEG (D SPE-PEG) . [0570] In certain embodiments the IDO inhibitor is conjugated to a moiety that forms a component of a vesicle structure in aqueous solution and is provided in the lipid bilayer (see, e.g., conjugated IDO inhibitors, supra.). In certain embodiments the IDO inhibitor is conjugated to a moiety such as a lipid, PHGP, vitamin E, cholesterol, and a fatty acid (see, e.g., Examples 1 and 2). In various embodiments the IDO inhibitor is conjugated directly to the vesicle-forming moiety and in other embodiments the IDO inhibitor is conjugated to the vesicle-forming moiety via a linker (e.g., via a homo-bifunctional or hetero-bifunctional linker). In certain embodiments the linker comprises an
Figure imgf000093_0001
linker.
[0571] In certain embodiments the inhibitor of the IDO pathway is conjugated to a lipid, and/or to PGHP and/or to vitamin E, and/or to cholesterol (CHOL), and/or to a fatty acid (e.g., oleic acid, docosahexaenoic acid, etc.). In certain embodiments the IDO inhibitor is conjugated to a lipid.
[0572] In certain embodiments the IDO inhibitor is conjugated to a phospholipid comprising said lipid bilayer or to cholesterol comprising said lipid bilayer. In certain embodiments the IDO inhibitor is conjugated to l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine (PL). In certain embodiments the IDO inhibitor conjugated to a phospholipid has the structure of IND-PL (see, e.g., Figure 26). [0573] In certain embodiments the ratio of phospholipid: CHOL:PEG, is about phospholipid (50-90 mol%): CHOL (10-50 mol%) : PEG (1-10 mol%). In certain
embodiments the lipid bilayer comprises IND-PL/Chol/DSPE-PEG. In certain embodiments as noted above, the bilayer comprises an IND-PL/Cholesterol/DSPE-PEG2K mixture at a molar ratio of 75:20:5. In certain embodiments the lipid bilayer is formulated to form a substantially uniform and intact bilayer encompassing the entire nanoparticle. In certain embodiments the lipid bilayer is formulated so that the mesoporous silica nanoparticle is colloidally stable.
Dual Delivery Lipid-Coated ICD-Inducing Nanomaterials.
[0574] It was discovered that certain nanomaterials are effective ICD inducers (see, e.g., Example 7). In certain embodiments these ICD-inducing nanomaterials can be administered simply as nanoparticles. However, in other embodiments, the nano particles can be combined with a lipid where the lipid is associated with (e.g., complexed with or conjugated to) an IDO pathway inhibitor (e.g., indoximod). In certain embodiments the lipid compires IND-PL (formula I). The lipid readily coats all or a part of the surface of the nanoparticle.
[0575] Accordingly in certain embodiments, a nanomaterial carrier for the combined delivery of an inhibitor of an IDO pathway and an inducer of immunogenic cell death (ICD), is provided wthere the nanomaterial carrier comprises a nanomaterial that induces ICD; and a lipid or lipid formulation comprising an IDO pathway inhibitor where the lipid or lipid formulation is disposed on the surface of said nanomaterial. In certain embodiments the lipid or lipid formulation fully encapsulates the nanomaterial, while in other embodiments, the lipid or lipid formulation is disposed on a surface of the nanoparticle, but does not fully encapsulate the nanoparticle. In certain embodiments the lipid or lipid formulation can form a lipid bilayer, while more typically, the lipid or lipid formulation is not a lipid bilayer.
[0576] In certain embodiments the ICD-inducing nanomaterial comprises one or more
ICD-inducing nanomaterials selected from the group consisting of CuO, Cu20, Sb203, As203, Bi203, P203, ZnO, Ti02, graphene oxide, 2D materials other than graphene or graphene oxide (e.g., graphene, graphyne, borophene, germanene, silicene, Si2BN, stanene, phosphorene, bismuthene, molybdenite, metals, 2D supracrystals, and the like) and other ICD-inducing nanomaterials as described herein. In certain embodiments the nanomaterial comprises copper oxide (CuO). In certain embodiments the nanomaterial comprises Sb203. In certain embodiments the nanomaterial comprises graphene oxide (GO).
[0577] In certain embodiments the IDO pathway inhibitor associated with the lipid or lipid formulation comprises an agent selected from the group consisting of 1 -methyl -D- tryptophan (indoximod, D-1MT), L-1MT, methylthiohydantoin-dl-tryptophan (MTH-Trp, Necrostatin), β-carbolines (e.g., 3-butyl-P-carboline), naphthoquinone-based (e.g., annulin- B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S- methyl-dithiocarbamate, S-hexyl-brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl- dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S[(naphth-2-yl)methyl]-dithiocarbamate, N-[2-
(indol-3-yl)ethyl]-S-[(pyrid-3-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid- 4-yl)methyl]-dithiocarbamate, 5-bromo-brassinin, Phenylimidazole-based IDO inhibitors (e.g., 4-phenylimidazole), Exiguamine A, imidodicarbonimidic diamide,N-methyl-N'-9- phenanthrenyl-monohydrochloride (NSC401366), INCB024360 (Epacadostat), 1- cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5-yl)ethanol (GDC-0919), IDO 1 -derived peptide, LG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl- 2-thiopseudourea hydrochloride, and 4-phenylimidazole. In certain embodiments the IDO pathway inhibitor associated with the lipid or lipid formulation comprises 1 methyl- tryptophan (1MT)). In certain embodiments the 1 methyl -tryptophan is a substantially pure "D" isomer of 1-methyl-tiyptophan (D-1MT), while in other embodiments, the 1-methyl- tryptophan is a substantially pure "L" isomer of 1 -methyl -tryptophan "L-1MT. In certain embodiments the 1-methyl-tiyptophan comprises a mixture of the D and L isomers.
[0578] In certain embodiments the IDO pathway inhibitor is conjugated to a lipid or to a component of the lipid formulation. In certain embodiments the IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid. In certain embodiments the IDO inhibitor is conjugated directly to the moiety, while in other emobodiments, the IDO inhibitor is conjugated to the moiety via a linker.
[0579] In certain embodiments the IDO pathway inhibitor is conjugated to PGHP, vitamin E, cholesterol (CHOL), a fatty acid, (e.g., oleic acid or docosahexaenoic acid), or to a lipid (e.g., a phospholipid). In certain embodiments the IDO pathway inhibitor is conjugated to a phospholipid. Illustrative phospholipids include, but are not limited to phospholipids comprising a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains. In certain embodiments the phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC),
dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), and diactylphosphatidylcholine (DAPC). In certain embodiments the phospholipid comprises a natural lipid selected from the group consisting of egg phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC). In certain embodiments the phospholipid comprises an unsaturated fatty acid selected from the group consisting of l,2-dimyristoleoyl-sn-glycero-3- phosphocholine, 1 ,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine, 1 ,2-dioleoyl-sn-glycero-3 - phosphocholine (DOPC), and l,2-dieicosenoyl-sn-glycero-3 -phosphocholine. In certain embodiments the phospholipid comprises l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine.
[0580] In certain embodiments the IDO pathway inhibitor comprises an agent selected from the group consisting of 1-methyl-D-tiyptophan (indoximod), 1-methyl-L- tryptophan, methylthiohydantoin-dl -tryptophan, Necrostatin-1, Ebselen, Pyridoxal
Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea
hydrochloride, Norharmane hydrochloride, INCB024360, S-allyl-brassinin, S-benzyl- brassinin, 5-Bromo-brassinin, 4-phenylimidazole Exiguamine A, and NSC401366. In certain embodiments the IDO pathway inhibitor comprises indoximod. In certain embodiments the IDO pathway inhibitor comprises substantially pure "L" isomer of 1-methyl-tiyptophan, or a substantially pure "D" isomer of 1-methyl-tiyptophan, or a racemic mixture of "D" and "L" isomers of 1 -methyl -tryptophan. In certain embodiments the 1 -methyl -tryptophan is conjugated to l-palmitoyl-2-hydroxy-5«-glycero-3 -phosphocholine (e.g., IND-PL, Formula I). Approach 2—Local treatment of a tumor or peritumor site to inhibit the IDO pathway and to induce ICD.
[0581] A second treatment modality involves local delivery to a tumor or peri- tumoral region, of an agent that induces ICD (e.g., doxirubicin, oxaliplatin, etc.) in combination with an inhibitor of the IDO pathway (e.g., indoximod). In certain
embodiments, the IDO inhibitor can be complexed with or conjugated to a moiety (e.g., a lipid) that forms a vesicle (e.g., a nanovesicle). It was discovered that such local delivery of an ICD inducer in combination with an IDO inhibitor induces recruitment of cytotoxic CD8+ lymphocytes, depletion of Tregs, reversal of the CD8+/Foxp3+ ratio, cytotoxic tumor killing, and tumor shrinkage at the local site. These adaptive immune responses were accompanied by boosting of the innate immune system, as reflected by CRT and HMGBl expression, as well as the activation of a DC population, particularly well-suited for generating cytotoxic T cell responses. [0582] Accordingly in certain embodiments, a method of treating a cancer in a mammal is provided where the method involves administering to an intra-tumoral or peritumoral site an effective amount of an inhibitor of the indoleamine 2, 3 -di oxygenase ( DO) pathway (an IDO inhibitor) in conjunction with an effective amount of an agent that induces immunogenic cell death (ICD) (an ICD-inducer). In certain embodiments, the effective amount of the ICD-inducer is an amount effective to elevate calreticulin (CRT) expression and/or to elevate expression and/or release of HMGBl and/or introduce ATP release in cells of the cancer.
[0583] ICD inducers are well known to those of skill in the art and ICD inducers suitable for this method will readily be recognized in view of the teachings provided herein. Illustrative ICD inducers include, but are not limited to chemotherapeutic agent(s) that induce ICD such as oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, oxaliplatin, paclitaxel, R2016 (a heterocyclic quinolone derivative described by Son et al. (2017) Plos One, DOI: 10.1371, which is incorporated herein by reference for the compounds described therein), and cyclophosphamide.
[0584] Other suitable ICD inducers include oncolytic viruses (see, e.g., Angel ova et al. (2014) J. Virol, 88(10): 5263-52760. One illustrative suitable oncolytic virus is an oncolytic parvovirus {e.g., H-PV).
[0585] As explained above, and in Example, 2, it was discovered that certain nanomaterials can induce ICD. In certain embodiments the ability to induce ICD is an intrinsic property of the nanomaterial {e.g., chemical reaction of the material and/or receptor binding of the nanomaterial is not required for induction of ICD). Accordingly, in certain embodiments the tumor or peritumoral space is treated with a nanomaterial that induces ICD. Such materials include, but are not limited to e.g., CuO, Cu20, Sb203, As203, Bi203, P203, ZnO, Ti02, graphene oxide, 2D materials other than graphene or graphene oxide {e.g., graphene, graphyne, borophene, germanene, silicene, Si2BN, stanene, phosphorene, bismuthene, molybdenite, metals, 2D supracrystals, etc.) and the like) {see, e.g., Example 2) nanoparticles comprising such materials. In certain embodiments the nanoparticle is entirely fabricated from said materials. In certain embodiments the nanoparticle comprises a doped material containing said materials. In certain embodiments the nanoparticle comprises a core-shell structure compmrising said ICD inducing materials. Accordingly, in certain embodiments ICD is induced by contacting the cancer cells with a nanomaterial (e.g., CuO, Sb203, ZnO, Ti02, and graphene oxide) that induced ICD.
[0586] It will also be recognized that in various embodiments, two or more ICD inducers can be used to induce ICD via local delivery.
[0587] In certain embodiments, the ICD inducer comprises at least oxaliplatin, or doxirubicin e.g., as described in Examples 3 and 4.
[0588] As noted above, the ICD inducer can be used in conjunction with an IDO inhibitor. Numerous IDO inhibitors are known to those of skill in the art (see, discussion below) and the use of one or more of these IDO inhibitors is contemplated. In certain embodiments the IDO inhibitor(s) comprise a conjugated IDO inhibitor as described herein. In certain embodiments the IDO inhibitors comprise indoximod or a conjugated indoximod as described below and in Examples 1 and 2. In certain embodiments the IDO inhibitors comprise substantially pure "D" indoximod, or substantially pure "L" indoximod, or conjugated substantially pure "D" indoximod, or conjugated substantially pure "L" indoximod. [0589] In certain embodiments the ICD inducer and the inhibitor of the IDO pathway are delivered locally to a target site. In certain embodiments the ICD inducer and the inhibitor of the IDO pathway can be delivered directly to a tumor site, e.g., by injection, or through a cannula. In certain embodiments the ICD inducer and the inhibitor of the IDO pathway are delivered into a tumor mass and/or into a peritumoral site. In certain
embodiments the ICD inducer and the inhibitor of the IDO pathway can be delivered as separate reagents. Alternatively, they can be delivered as a combined formulation. In certain embodiments the combined formulation comprise nanovesicles and/or lipid bilayer coated silica nanoparticles, e.g. as described herein, or suitable other dual delivery carriers that contain an IDO inhibitor plus a nanomaterial capable of inducing ICD. [0590] In certain embodiments the ICD inducer and the IDO pathway inhibitor are delivered via an implantable depot delivery system (e.g., encapsulated in a controlled release polymer, a hydrogel, and the like). In certain embodiments both the ICD inducer and the the IDO pathway inhibitor are in implantable depot delivery systems and in other embodiments only the the IDO pathway inhibitor or the ICD inducer is in an implantable depot delivery system.
[0591] In certain embodiments the ICD inducer and the IDO pathway inhibitor are used in combination as a primary therapy. In certain embodiments the ICD inducer and the IDO pathway inhibitor are used as an adjunct therapy, e.g., in combination with other chemotherapeutics, and/or surgery, and/or radio therapy. In certain embodiments the ICD inducer and the the IDO pathway inhibitor are delivered to a surgical site during or after removal of a tumor mass. [0592] In view of the examples and teachings provided herein, it will be recognized that the co-delivery of an ICD inducer and the IDO pathway inhibitor will find use in the treatment of a number of cancers. Illustrative cancers include, but are not limited to pancreatic ductal adenocarcinoma (PDAC), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Adrenocortical carcinoma, Kaposi sarcoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors,
craniopharyngioma, ependymoma, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, kaposi sarcoma, kidney cancer (e.g., renal cell, Wilm's tumor, and other kidney tumors), langerhans cell histiocytosis, laryngeal cancer, leukemia, acute
lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myelogenous (CML), hairy cell, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer {e.g., childhood, non-small cell, small cell), lymphoma {e.g., AIDS-related, Burkitt {e.g., non-Hodgkin lymphoma), cutaneous T-Cell {e.g., mycosis fungoides, Sezary syndrome), Hodgkin, non-Hodgkin, primary central nervous system (CNS)), macroglobulinemia, Waldenstrom, male breast cancer, malignant fibrous
histiocytoma of bone and osteosarcoma, melanoma {e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple
myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes,
Myelogenous Leukemia, Chronic (CML), multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma {e.g., Ewing,
Kaposi, osteosarcoma, rhadomyosarcoma, soft tissue, uterine), Sezary syndrome, skin cancer {e.g., melanoma, merkel cell carcinoma, basal cell carcinoma, nonmelanoma), small intestine cancer, squamous cell carcinoma, squamous neck cancer with occult primary, stomach (gastric) cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom
macroglobulinemia, and Wilm's tumor.
[0593] In certain embodiments the cancer to be treated is cancer pancreatic ductal adenocarcinoma (PDAC) and in certain embodiments, the ICD inducer comprises oxaliplatin and the IDO inhibitor comprises indoximod or a conjugated indoximod as described below in in Example 1. Approach 3— Vaccination to prevent or treat a cancer,
[0594] In various embodiments, methods are provided for the prevention or treatment of a cancer that involve vaccinating a subject (e.g., a human, or a non-human mammal) to induce an immune response directed against one or more cancers. It was a surprising discovery that vaccination of a mammal with cancer cells in which ICD has been induced ex vivo is sufficient to generate a systemic immune response that can interfere with tumor growth at a remote site as well as allowing adoptive transfer to non-immune animals.
[0595] Without being bound to a particular theory it is believed that such vaccination methods can be used for the treatment of an existing cancer or prophylactically to prevent or inhibit the formation of a cancer in a subject. In the latter case, for example, subjects that have a family history for cancer in general or for particular cancers, and/or that have a genetic risk for a cancer (e.g., mutations in BRCA1, and/or BRCA2, and/or P53) may be vaccinated prophylactically to prevent the development of a cancer.
[0596] In certain embodiments, the vaccination is used as a primary therapy in the treatment of a cancer. In certain embodiments the vaccination is used as an adjunct therapy, e.g., in combination with surgery, and/or other chemotherapy regimen, and/or radiation therapy.
[0597] Accordingly, in various embodiments, a method for the treatment and/or prevention of a cancer in a mammal is provided where the method comprises providing cancer cells in which immunogenic cell death (ICD) has been induced ex vivo, and vaccinating the mammal with these cells, where the vaccination induces an anti-cancer immunogenic response.
[0598] In certain embodiments, the cancer cells are cells derived from an existing cancer, e.g., obtained during a biopsy, or after surgical resection of a tumor mass). In certain embodiments the cancer cells are cells obtained from the subject that is to be treated and comprise an autologous transplant. In certain embodiments the cells are obtained from a different subject of the same species or can even be obtained from a different species.
[0599] In certain embodiments, the cancer cells are cells from a cancer cell line.
Typically, where a non-human animal is to be treated (veterinary use) the cell line is an animal cell line from the same species that is to be treated. Similalry, where a human is to be treated a human cell line will typically be used. Numerous cancer cell lines are known to those of skill in the art. Illustrative, but non-limiting examples of suitable cell lines are shown in Table 1.
Table 1. Illustrative, but non-limiting, cell lines that can be used to produce dying cancer cells in which immunogenic cell death (ICD) has been induced.
Figure imgf000102_0001
EoL-1 cell Human eosinophilic leukaemia
VCaP Human Prostate Cancer Metastasis
tsA201 Human embryonal kidney, SV40 transformed
HT 1080 Human fibrosarcoma
PANC-1 Human Caucasian pancreas
Saos-2 Human primary osteogenic sarcoma
ATCC Pancreatic cell lines:
Capan-2 ATCC HTB-80
Pane 10.05 ATCC CRL-2547
CFPAC-1 ATCC CRL-1918
HPAF-II ATCC CRL-1997
SW 1990 ATCC CRL-2172
BxPC-3 ATCC CRL-1687
AsPC-1 ATCC CRL-1682
ATCC colon cancer lines
SNU-C1 ATCC CRL-5972
SK-CO-1 ATCC HTB-39
SW1116 ATCC CCL-233
SW948 ATCC CCL-237
T84 ATCC CCL-248
LS123 ATCC CCL-255
LoVo ATCC CCL-229
SW837 ATCC CCL-235
SNU-C1 ATCC CRL-5972
SW48 ATCC CCL-231
RKO ATCC CRL-2577
COLO 205 ATCC CCL-222
SW1417 ATCC CCL-238
LS411N ATCC CRL-2159
NCI-H508 ATCC CCL-253
HT-29 ATCC HTB-38
CRL-1718™ CCF-STTG1 Human Brain Astrocytoma
HTB-12™ SW 1088 Human Brain Astrocytoma
HTB-13™ SW 1783 Human Brain Astrocytoma
CRL-3020™ CHLA-02- Human Brain Atypical Teratoid Rhabdoid Tumor (ATRT)
ATRT
CRL-1620™ A 172 Human Brain Glioblastoma
HTB-16™U-138 MG Human Brain Glioblastoma
CRL-2610™LN-18 Human Brain Glioblastoma
CRL-2611™LN-229 Human Brain Glioblastoma
HTB-14™U-87 MG Human Brain Glioblastoma, astrocytoma
HTB-15™U-118 MG Human Brain Glioblastoma, astrocytoma
CRL-1690™ T98G Human Brain Glioblastoma, multiforme
HTB-138™Hs 683 Human Brain Glioma
CRL-3021™CHLA-01- Human Brain Medullomyoblastoma
MED
CRL-2273™CHP-212 Human Brain Neuroblastoma
HTB-148™H4 Human Brain Neuroglioma HTB-187™D341 Med Human Brain, cerebellum Medulloblastoma
HTB-186™Daoy Human Brain, cerebellum Medulloblastoma, desmoplastic cerebellar
CRL-2060™PFSK-1 Human Brain, cerebellum Tumor, malignant primitive
neuroectodermal
CRL-2020™ DBTRG- Human Brain, glial cell Glioblastoma
05MG
CRL-2365™M059K Human Brain, glial cell Glioblastoma
CRL-2366™ M059J Human malignant glioblastoma
[0600] Although not required, in typical embodiments the cancer cells used in the vaccination are of the same type of cancer that is to be treated and/or prevented. It will be recognized however, that vaccination with cells of one type of cancer may generate an immune response directed against a different cancer and/or against multiple cancers. In certain embodiments the vaccination is with cells from multiple different types (e.g., 2 or more cancers, 3 or more cancers, 4 or more cancers, 5 or more cancers, 6 or more cancers, 7 or more cancers, 8 or more cancers, 9 or more cancers, 10 or more cancers, etc.) in which ICD is induced.
[0601] In certain embodiments illustrative cancers to be treated or prevented include, but are not limited to pancreatic ductal adenocarcinoma (PDAC), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Adrenocortical carcinoma, Kaposi sarcoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, kaposi sarcoma, kidney cancer (e.g., renal cell, Wilm's tumor, and other kidney tumors), langerhans cell histiocytosis, laryngeal cancer, leukemia, acute
lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myelogenous (CML), hairy cell, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., childhood, non-small cell, small cell), lymphoma (e.g., AIDS-related, Burkitt (e.g., non-Hodgkin lymphoma), cutaneous T-Cell (e.g., mycosis fungoides, Sezary syndrome), Hodgkin, non-Hodgkin, primary central nervous system (CNS)), macroglobulinemia, Waldenstrom, male breast cancer, malignant fibrous
histiocytoma of bone and osteosarcoma, melanoma (e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple
myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes,
Myelogenous Leukemia, Chronic (CML), multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Ewing, Kaposi, osteosarcoma, rhadomyosarcoma, soft tissue, uterine), Sezary syndrome, skin cancer (e.g., melanoma, merkel cell carcinoma, basal cell carcinoma, nonmelanoma), small intestine cancer, squamous cell carcinoma, squamous neck cancer with occult primary, stomach (gastric) cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom
macroglobulinemia, and Wilm's tumor.
[0602] In various embodiments the cells used in the vaccination include cells of one or more of these cancers. [0603] Methods of inducing immunogenic cell death (ICD) are well known to those of skill in the art. In certain embodiments ICD is induced by contacting the cells (e.g., primary tumor cells, cancer cell lines, etc.) with one or more chemotherapeutic agent(s) that induce ICD. Such agents include, but are not limited to oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016 (a heterocyclic quinolone derivative described by Son et al. (2017) Plos One, DOL 10.1371, which is incorporated herein by reference for the compounds described therein), and cyclophosphamide. In certain embodiments the ICD chemo reagents may also include the drug derivatives, i.e. prodrugs, that are capable of releasing the abovementioned chemotherapeutics in biological environments.
[0604] Another method of inducing ICD involves infecting the cells with an oncolytic virus. Illustrative, but non-limiting oncoviruses that induce ICD include, but are not limited to Parvovirus (e.g., H-PV (see, e.g., Angelova et al. (2014) J. Virol, 88(10): 5263-5276), and the like), Adenovirus (AD) (e.g., hTERT-Ad (see, e.g., Boozari et al. (2010) Gut. 59: 1416- 1426), Ad5/3-D24-GMCSF (see, e.g., Liikanen et al. (2013) Mol. Ther. 21 : 1212-1223), and the li.ke), Herpes simplex virus (HSV) (e.g., G207 (see, e.g., Toda et al. (1999) Hum. Gene. Ther. 10: 385-393), HSV-1716 (see, e.g., Benencia et al. (2005) Mol. Ther., 12: 789-8020, T- VEC (see, e.g., Hu et al. (2006) Clin. Cancer Res. 12: 6737-67470), HSV-2 ΔΡΚ mutant (see, e.g., Colunga et al. (2010) Gene Ther., 17: 315-327), and the like), Poxvirus (e.g., vSP (see, e.g.,Guo et al. (2005) Cancer Res. 65: 9991-9998, vvDD (see, e.g., John et al. (2012) Cancer Res., 72: 1651-1660), Pexa-Vec (see, e.g., Heo et al. (2013) Nat. Med, 19: 329-336), and the like), Arbovirus (see, e.g., VSV-GFP (Indiana serotype) (see, e.g., Wongthida et al. (2010) Cancer Res. 70: 4539-4549), VSVgm-icv (see, e.g., Lemay et al. (2012) Mol. Ther., 20: 1791-1799), and the like), Paramyxovirus (e.g., MV-eGFP (Edmonston strain) (see, e.g., Donnelly et al. (2013) Gene Ther. 20: 7-15), and the like). A review of such oncoviruses is found in Bartlett et al. (2013) Mol. Cancer. 12: 103).
[0605] Other methods of inducing ICD involve exposure to radiation (e.g., gamma radiation, UVC radiation).
[0606] In certain embodiments ICD induction is accomplished using any of the compounds and/or modalities described in Table 2.
Table 2. Illustrative compounds and/or modalities to induce immunogenic cell death (ICD).
Figure imgf000106_0001
Oxaliplatin apoptotic secreted ATP; mid to late
UVC irradiation apoptotic ecto-HSP70; late apoptotic γ-irradiation passively released HMGB 1
anthracyclines (e.g., Daunorubicin,
Doxorubicin, Epirubicin, Idarubicin)
Early to mid apoptotic ecto-CRT; early to mid apoptotic ecto-HSP70; early to mid
Shikonin
apoptotic ecto-GRP78
Pre-apoptotic ecto-CRT and ERp57; early
7A7 (EGFR-specific antibody) to mid apoptotic ecto-HSP70; early to mid apoptotic ecto-HSP90
Pre-apoptotic ecto-CRT; late apoptotic
Cyclophosphamide passively released HMGB 1
Bortezomib Early to mid apoptotic ecto-HSP90
Pre-apoptotic ecto-CRT; early to mid
Cardiac glycosides apoptotic ATP release; late apoptotic
passively released HMGB 1
Pre-apoptotic ecto-CRT; pre-apoptotic secreted ATP; pre-apoptotic ecto-HSP70;
Hypericin-based PDT
tate apoptotic passively released HSP70, HSP90 and CRT
Early apoptotic ecto-CRT; early apoptotic
Coxsackievirus B3 secreted ATP; late apoptotic passively
released HMGB 1
Oncolytic parvovirus (e.g., H-PV)
Anthracenedi one
Bleomycin
Docetaxel
Paclitaxel
R2016
[0607] In other embodiments, the methods of inducing ICD can involve contacting the cells with materials, e.g., nanomaterials that induce ICD. It was a surprising discovery that certain materials (e.g., nanomaterials), as a result of intrinsic nanomaterial properties, are capable of inducing ICD, e.g., as determined by CRT induction, in a manner comparable to the positive control, oxaliplatin. Such materials include, but are not limited to CuO, Sb203, ZnO, Ti02, and graphene oxide (see, e.g., Example 3). Accordingly, in certain embodiments ICD is induced by contacting the cancer cells with a nanomaterial that induces ICD (e.g., CuO, Cu20, Sb203, As203, Bi203, P203, ZnO, Ti02, graphene oxide, 2D materials other than graphene or graphene oxide (e.g., graphene, graphyne, borophene, germanene, silicene, Si2BN, stanene, phosphorene, bismuthene, molybdenite, metals, 2D supracrystals, and the like)). In certain embodiments, the nanomaterial comprises copper oxide. In certain embodiments, the nanomaterial comprises Sb203. In certain embodiments, the nanomaterial comprises graphene oxide (GO). CuO, Cu20, Sb203, As203, Bi203, P203, ZnO, Ti02, graphene oxide, and 2D materials other than graphene or graphene oxide
[0608] Extensive high throughput screening of a large number of nanomaterial libraries (including metals, metal oxides, rare earth oxides, graphene, graphene oxide, multi- and single walled carbon nanotubes, fumed silica, long aspect ratio nanomaterials, redox active nanomaterials, nanomaterials with functionalized catalytic surfaces and coatings etc) in our nanomaterial safety screening laboratory in the California Nano Systems Institute at UCLA have demonstrated a variety of mechanisms, involving intrinsic nanomaterial properties, that can induce a wide variety of different types of cell death, including apoptosis, necrosis, pyroptosis, and immunogenic cell death.
[0609] In view of the ICD-inducing properties of graphene oxide (GO), it is believed that numerous other 2-dimensional (2D) materials can similarly induce ICD.
[0610] A number of 2D materials other than graphene are known to those of skill in the art {see, e.g., Mas Balleste et al. (2011) Nanoscale, 3 : 20-30). Such materials include, but are not limited to graphene, graphyne, borophene, germanene, silicene, Si2BN, stanene, phosphorene, bismuthene, molybdenite, metals, 2D supracrystals, and the like. Other 2D materials include, but are not limited to BN, MoS2, NbSe2, Bi2Sr2CaCu20x (Id.), single layers of single layers of manganese (see, e.g., Omomo et al. (2003) J. Am. Chem. Soc, 125: 3568- 3575), oxides of cobalt (see, e.g., Kim et al. (2009) Chem. Eur. J, 15: 10752-10761), tantalum (Fukuda et al. (2007) Inorg. Chem. 46: 4787-4789), ruthenium (Fukuda et al. (2010) Inorg. Chem. 49: 4391-4393), and titanium (Tanaka et a/. (2003) Chem. Mater. 15: 3564- 3568) as well as sheets of several perovskite type structures, e.g., H2[An-iBn03n+i] where A is Na, CA, Sr, or LA, and B is Ta or Ti, K2LN2Ti3Oi0A, KLnNb207, or RbLNTa207 where Ln is lanthanide ion, MW06 where M is Nb or Ta, KCa2 b3Oi0, KSr2Nb3Oi0, Bi2SrTa209, and the like.
[0611] It is noted that these ICD-inducing nanomaterials exhibit a range of tunable physicochemical properties that can readily be adapted to achieve the optimal ICD-inducing catalytic outcomes. For example, for graphene oxide these properties include, inter alia, nanosheet size, surface oxidation status, and the like, while for metal oxides these properties include, inter alia, the particle size, dissolution characteristics, zeta potential, and the like.
[0612] The list of nanomaterials above that induce immunogenic cell death is illustrative and non-limiting. It is believed there are numerous other materials that have the capability of inducing ICD based on property-activity relationships, such as the induction of oxidative stress, mitochondrial damage, lysosomal damage, surface membrane damage, DNA damage, photo activation, oxygen radical generation, activation of the NRLP3
inflammasome, induction or interference in autophagy flux, etc.
[0613] It will also be recognized that in various embodiments, two or more agents can (e-g-, two or more of the agents or modalities described above) can be used to induce ICD.
[0614] Methods of determining whether IDC is induced in the cells are known to those of skill in the art. For example, ICD is characterized by elevated expression of calreticulin (CRT), and/or elevated expression and/or release of e.g., HMGB1 or ATP as compared to the same cells in which ICD is not induced. Illustrative, but non-limiting methods of inducing ICD in cancer cells (e.g., KPC cells) and evaluation of the ICD are described in Example 1.
[0615] These methods and agents for inducing ICD are illustrative and non-limiting.
Numerous other agents and compositions for inducing ICD are known.
Modes of vaccination.
[0616] Methods of vaccination of humans or non-human mammals are well known to those of skill in the art. Most typically, the vaccination will be by intramuscular,
subcutaneous, or intradermal injection. In various embodiments injection may be performed by needle or pressure.
[0617] In certain embodiments mucosal immunization can be performed and such modalities include, but are not limited to intraocular, intranasal and/or oral.
[0618] In certain embodiments jet injectors, such as Antares Pharma's MediJector
VISION, deliver medication through high-speed, pressurized liquid penetration of the skin without a needle. These have been developed as single-use devices and multiuse systems. A high peak pressure behind the liquid is required so it can drill a hole in the skin, and then the pressure is reduced to allow the rest of the liquid to enter the skin. [0619] Other transdermal approaches deliver the antigen in a solid form. These approaches have the added benefit that the therapeutic agent is more stable and therefore may not need cold storage.
[0620] Another illustrative, but non-limiting approach uses the pharmaceutical formulation itself to puncture the skin. Glide Pharma has developed a low-velocity, spring- powered administrator that pushes a pointed rod of pharmaceutical material through the skin in a fraction of a second. This administrator enables constant, reliable delivery of a solid dosage form and could be applied to various vaccines including vaccines comprising cancer ICD-induced cancer cells as described herein. [0621] In another illustrative, but non-limiting embodiment, the antigen (e.g., ICD- induced cancer cells) can be delivered by injection or implantation in a hydrogel. In certain embodiments the hydrogel is an injectible hydrogel.
[0622] Injectable hydrogels can be prepared using a wide range of materials. Cyto- and bio-compatibility as well as reactive chemistries are typical factors considered for selecting base materials that can be used in hydrogels for cell delivery. Material crosslinking (formation and concentration of physical or covalent linkages), biodegradability, and biochemical properties can influence the structural, mechanical, and biological properties of the hydrogels initially and over time. Hydrophilic polymers used for hydrogel construction generally can be divided into two categories: natural polymers derived from tissues or other natural sources and synthetic polymers fabricated using organic chemistry and molecular engineering principles. Biocompatible natural polymers such as hyaluronic acid, chitosan, heparin, alginate, fibrin, collagen, chondroitin sulfate, and silk, mimic aspects of the native microenvironment, including its mechanical and biochemical properties for modulating cell adhesion, migration, and other functions (see, e.g., Munarin et al. (2012) J. Appl. Biomater. Funct. Mater. 10(2): e67-81). These natural polymers have been used as building blocks for injectable hydrogel formation by physical (e.g., ionic, hydrogen bonding) or covalent crosslinking (e.g., reaction of functional groups on modified polymers) (see, e.g., Kharkar et al. (2013) Chem. .Soc. Rev. 42(17): 7335-7372.
[0623] Synthetic polymers such as poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(N-isopropylacrylamide) (PNIPAAm), and polycaprolactone (PCL) have frequently been used for the design of injectable, cell-compatible hydrogels due to their commercial availability, low batch-to-batch variation, versatility for chemical modification, and consequently, the ease of tuning the mechanical properties of the resulting hydrogels. Since synthetic polymers lack the inherent biochemical cues for interaction with cells, In certain embodiments they can be used in combination with natural polymers or biomimetic peptides to facilitate cell adhesion, migration, and protein secretion. [0624] In certain illustrative, but non-limiting embodiments, the cells can be delivered by use of an injectable (or implantable) cryogel. Cryogels are a type of hydrogel made up of cross-linked hydrophilic polymer chains that can hold up to 99 percent water. They are created by freezing a solution of the polymer that is in the process of gelling. When thawed back again to room temperature, the substance turns into a highly interconnected pore- containing hydrogel, which is similar in composition to bodily soft tissues in terms of their water content, structure, and mechanics. By adjusting their shape, physical properties, and chemical composition sponge-like, porous cryogels can be formed that can be infused with living cells, biological molecules or drugs. One illustrative, but non-limiting cyrogel is formed from methacrylated alginate (MA-alginate) as described by Bencherif et al. (2016) Nat. Comm., 6: 7556.
Adjuvants.
[0625] In certain embodiments the vaccination utilizing cancer cells in which ICD has been induced is performed using one or more adjuvants to increase the subject's immune response to the vaccination. Typically, adjuvants enhance and direct the adaptive immune response to vaccine antigens.
[0626] Adjuvants may exert their effects through different mechanisms. Some adjuvants, such as alum and emulsions {e.g., MF59®), function as delivery systems by generating depots that trap antigens at the injection site, providing slow release in order to continue the stimulation of the immune system. These adjuvants enhance the antigen persistence at the injection site and increase recruitment and activation of antigen presenting cells (APCs). Particulate adjuvants {e.g., alum) have the capability to bind antigens to form multi-molecular aggregates that encourage uptake by APCs {see, e.g., Leroux-Roels (2010) Vaccine. 288(3) :C25-3).
[0627] Some adjuvants are also capable of directing antigen presentation by the major histocompatibility complexes (MHC) {Id.). Other adjuvants, essentially ligands for pattern recognition receptors (PRR), act by inducing the innate immunity, predominantly targeting the APCs and consequently influencing the adaptive immune response. AIOOH described below is one such example. Members of nearly all of the PRR families are potential targets for adjuvants. These include Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I- like receptors (RLRs) and C-type lectin receptors (CLRs). They signal through pathways that involve distinct adaptor molecules leading to the activation of different transcription factors. These transcription factors (e.g., NF-κΒ, IRF3) induce the production of cytokines and chemokines that play a key role in the priming, expansion and polarization of the immune responses. Activation of some members of the NLR family, such as NLRP3 and NLRC4, triggers the formation of a protein complex, called inflammasome, implicated in the induction of the pro-inflammatory cytokines IL-Ιβ (see, e.g., Li et al. (2008) J. Immunol. 181(1): 17- 21.) and IL-18. The NLRP3 and NLRC4 inflammasomes have been involved in the innate immunity induced by certain adjuvants. Much of our high throughput discovery on material such as AOOH, multiwall carbon nanotubes, singlewall carbon nanotubes, graphene, rare earth oxide nanoparticles, metal oxide nanorods, and the like function via the NRLP3 pathway. Most of the adjuvant effects fit into the category of stimulating DAMP pathways, which overlaps with the concept of ICD.
Alum & emulsions
[0628] Alum is the most commonly used adjuvant in human vaccination. Alum provokes a strong Th2 response. Alum induces the immune response by a depot effect and activation of APCs. The NLRP3 inflammasome has been linked to the immunostimulatory properties of alum.
[0629] In certain embodiments a high aspect ratio AIOOH variant of alum can be used as an adjuvant. We have also made a much-improved variant of alum by high throughput screening that identified high aspect ration AIOOH for use as an adjuvant. The high aspect ratio AIOOH that is 1-2 orders of magnitude better than Alum, based, inter alia, on the principle that the long aspect ratio of the material and its surface reactivity provide superior stimulation to the NRLP3 inflammasome in dendritic cells (see, e.g., Sun et al.
(2013) ACS Nano, 7(12): 10834-10849).
[0630] Additionally, emulsions (either oil-in-water or water-in-oil), such as Freund's
Incomplete Adjuvant (IF A) and MF59®, can trigger depot generation and induction of MHC responses. IFA induces a predominantly Th2 biased response with some Thl cellular response. MF59® is a potent stimulator of both cellular (Thl) and humoral (Th2) immune responses. PRR Ligands
[0631] New adjuvants are being developed that are natural ligands or synthetic agonists for PRRs, either alone or with various formulations. PRR activation stimulates the production of pro-inflammatory cytokines/chemokines and type I IFNs that increase the host's ability to eliminate the pathogen. Thus, the incorporation of pathogens associated molecular patterns (PAMPs) in vaccine formulations can improve and accelerate the induction of vaccine-specific responses. A number of these agonists are now in clinical or late preclinical stages of development (see, e.g., Steinhagen et al. (2011) 29(17): 3341-3355; Mbow et al. (2010) Curr. Opin. Immunol. 22(3): 411-416). When used in combination with alum or classical emulsion adjuvants, the immune response can be biased towards a Thl response (see, e.g., Didierlaurent et al. (2009) J. Immunol. 183(10): 6186-6197).
TLR3 and RLR Ligands
[0632] Double-stranded RNA (dsRNA), which is produced during the replication of most viruses, is a potent inducer of innate immunity. Synthetic analogs of dsRNA, such as poly(LC), have been utilized as adjuvants. They act through TLR 3 and RIG-I/MDA-5, inducing IL-12 and type I IFNs production, facilitating antigen cross-presentation to MHC class II molecules, and improving generation of cytotoxic T cells.
TLR4 Ligands
[0633] Bacterial lipopolysaccharides (LPS), which are ligands for TLR4, have long been recognized as potent adjuvants. The development of less toxic derivatives led to the production of MPLA (monophosphoryl lipid A), which formulated with alum (AS04) triggers a polarized Thl response and is approved for clinical use in Europe. We also have demonstrated that graphene oxide can interact with TLR4.
TLR5 Ligands
[0634] The TLR5 ligand, bacterial flagellin, is a potent T-cell antigen and has been utilized as a vaccine adjuvant. Unlike other TLR agonists, flagellin tends to produce mixed Thl and Th2 responses rather than strongly Thl responses. Flagellin can be used as an adjuvant mixed with the antigen.
-I l l- TLR7/8 Ligands
[0635] The TLR7/8 pathway, specialized in the recognition of single stranded viral
RNA, has also been explored for use as vaccine adjuvants. Imidazoquinolines (e.g., imiquimod, gardiquimod, and R848) are synthetic compounds that activate TLR7/8 in multiple subsets of dendritic cells leading to the production of IFN-a and IL-12 thus promoting a Thl response. In this regard, is is noted that the formulations and/or drug delivery nanocarriers described herein can can easily include imiquimod.
TLR9 Ligands
[0636] Oligodeoxynucleotides containing specific CpG motifs (CpG ODNs such as ODN 1826 and ODN 2006) are recognized by TLR9. They enhance antibody production and strongly polarize the cell responses to Thl and away from Th2 responses. In this regard, it is noted that various a drug delivery nanocarriers described herein (e.g., a bilayer-coated nanoparticle) can readily be modified to present CPG oligonucleotides on the surface (e.g., LB-coated nanoparticles can present CPG oligo's on the lipid bilayer). NOD2 Ligands
[0637] Fragments of bacterial cell walls, such as muramyl dipeptide (MDP), have long been recognized as adjuvants. More recently, it was discovered that MDP triggers the activation of NOD2 and the NLRP3 inflammasome.
[0638] Adjuvants may be combined to achieve a stronger effect or a more potent skewing of immune responses. For example, alum has been combined with TLR9 agonists (see, e.g., Siegrist et al. (2004) Vaccine, 23(5): 615-622). In experimental models, administration of other combinations such as CpG ODNs with MDP or MPLA has proven effective (see, e.g., Kim et al. (2000) Vaccine, 19: 530-537).
[0639] In various embodiments, any one or more of the these adjuvants may be used to enhance response to the vaccination with cancer cells in which ICD has been induced.
[0640] The foregoing vaccination methods are illustrative and non-limiting. Using the teachings provided herein, numerous other methods and compositions for vaccinating subjects with cancer cells in which ICD is induced will be available to one of skill in the art. IDO inhibitors
[0641] A number of IDO inhibitors are well-known to those of skill in the art and useful in the methods described herein. Illustrative, but non-limiting examples of IDO inhibitors are shown in Table 3 and the structures of several of these are shown in Figure 2. Table 3. Illustrative, but non-limiting IDO inhibitors.
Figure imgf000115_0001
Figure imgf000116_0001
y roc o e
[0642] Still other IDO inhibitors include, but are not limited to the inhibitors described in U.S. Patent Publication Nos: US 2016/0362412, US 2016/0289171, US
2016/0200674, US 2016/0143870, US 2016/0137595, US 2016/0060237, US 2016/0002249, US 2014/0323740, US 2014/0066625, US 2013/0289083, US 2013/0183388, US
2012/0277217, US 2011/0136796, US 2011/0112282, US 2011/0053941, US 2010/0233166, US 2010/0166881, US 2010/0076066, US 2009/0042868, US 2007/0173524, US
2007/0105907, which are all incorporated herein by reference for the IDO inhibitors described therein. [0643] It is contemplated that the methods described herein can use one or more of these IDO inhibitors and/or any other IDO inhibitors known to those of skill in the art. In certain embodiments the one or more IDO inhibitors comprise indoximod.
Conjugated IDO inhibitors and vesicles thereof.
[0644] In certain embodiments one or more IDO inhibitors (e.g., any one or more of the IDO inhibitors shown in Table 3) are conjugated to a moiety that forms a vesicle (e.g., a liposome) structure in aqueous solution or that can form a component of a lipid bilayer comprising a liposome. The conjugated IDO inhibitors can be used directly (e.g., described in approach 2 above), provided as components in a combined formulation (e.g., in combination with an ICD inducer), and in certain embodiments, the IDO inhibitor is conjugated to a moiety that forms a component of a lipid bilayer that can be disposed on a nanoparticle, e.g., as described below and in Example 1).
[0645] In certain embodiments the moiety that is conjugated to the the IDO pathway inhibitor comprises a lipid, PHGP, vitamin E, cholesterol, and/or a fatty acid. In certain embodiments the IDO pathway inhibitor can be conjugated directly to the moiety (see, e.g., Figure 3), while in other embodiments the IDO inhibitor can be conjugated to the moiety using a linker (e.g., a HO-(CH2)n=2-5-OH linker as shown in Figure 4).
[0646] In the illustrative embodiments shown in Figure 3, the an ester bond is used to make the conjugate. As a general strategy in the case of indoximod, the H2 group in the indoximod is protected before the conjugation reaction. The -COOH in indoximod can then robustly react with the in the conjugating moiety (e.g., PHGP, Vitamin E, cholesterol, a fatty acid, a lipid, etc.). Similarly, Figure 54 illustrates representative examples to show the combined use of HO-(CH2)n=2-5-OH linker and ester bond to make IDO inhibitor (e.g., indoximod) pro-drug conjugates. Again, as illustrated, the NH2 group can be protected. [0647] These reactions, however, are illustrative and non-limiting. Numerous IDO inhibitors have other groups readily available for conjugation directly to a vesicle-forming moiety or to a linker. Such groups include for example, H, OH, CH2, and the like (see, e.g., Figure 2).
[0648] In certain embodiments, particularly for rapid and easy incorporation into a lipid bilayer the IDO pathway inhibitor can be conjugated to a lipid (e.g., a phospholipid), or cholesterol. Of course, in certain embodiments, the other vesicle-forming agents having conjugated IDO inhibitor(s) can also be incorporated into a lipid bilayer. [0649] In certain embodiments, the inhibitor of the IDO pathway is conjugated to a phospholipid comprising a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains. In certain embodiments, the
phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC),
distearoylphosphatidylcholine (DSPC), and diactylphosphatidylcholine (DAPC). In certain embodiments, the phospholipid comprises a natural lipid selected from the group consisting of egg phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC). In certain embodiments, the phospholipid comprises an unsaturated fatty acid selected from the group consisting of l,2-dimyristoleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoleoyl-sn- glycero-3-phosphocholine,l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2- dieicosenoyl-sn-glycero-3-phosphocholine.
[0650] In certain embodiments, the phospholipid comprises l-palmitoyl-2-hydroxy- sn-glycero-3-phosphocholine. In certain embodiments, the lipid conjugated inhibitor of the IDO pathway comprises l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine conjugated to 1-methyl-tryptophan, and in certain embodiments the lipid conjugated inhibitor comprises IND-PL having the structure shown in Formula I:
Figure imgf000118_0001
[0651] It will be recognized that as shown above, in various embodiments, the 1- methyl-tryptophan component of IND-PL can be a "D" isomer or an L isomer.
[0652] In certain embodiments the IDO pathway inhibitors can be incorporated into the lipid bilayer forming the vesicle witout conjugation to a lipid bilayer component. For example, epacadostat is a potent direct IDO enzyme inhibitor with an IC50 of -125 nM in a whole blood assay (Yue et al. (2017) ACS Med. Chem. Letts. 8: 486-491). Although the drug showed good synergy with anti-PDl antibody (nivolumab) in a phase II clinical trial in melanoma patients, the success could not be duplicated in a recent phase 3 clinical trial for the same disease. This has raised questions about the exact role and efficacy of IDO inhibitors, their pharmacology and explaining the divergent effects. Epacadostat is highly soluble in ethanol (>20 mg/mL), which allows its incorporation into a liposomal membrane through the use of the ethanol injection method {{see, e.g., Pons, et al. (1993) J.
Pharmaceutic. 95: 51-56). The ethanol injection method produces homogeneous unilamellar liposomes (Pereira et al. (2016) Int. J. Pharmaceutics, 514: 150-159). In this method, water is poured into a concentrated lipid-ethanol solution (containing docetaxel and possibly IND- PL in a ratiometric designed strategy), following which ethanol is removed in an evaporator (Id.). Dilution with water causes spontaneous formation of small and homogenous unilamellar liposomes from the micellar aggregate. The size of the liposomes can be controlled by the ratio of ethanol to water. [0653] It will be recognized that the foregoing conjugates and lipid bilayers incorporating IDO inhibitors are illustrative and non-limiting. Using the teaching provided herein, numerous other IDO inhibitor conjugates and/ro IDO-containign lipid bilayers (vesicles) will be available to one of skill in the art.
Remote loading of Silicasomes, and Vesicles
[0654] In certain embodiments encapsulation of, e.g., the ICD inducer in the nanoparticle and/or in the nanovesicle is optimized by using a "remote loading" strategy in which the addition of the drug {e.g., ICD-inducer such as doxorubicin) to preformed vesicles or silicasomes (LB-coated nanoparticles) which achieves high loading levels using a a pH gradient or an ion gradient capable of generating a pH gradient (see, e.g., Ogawa et al. (2009) J. Control. Pel. 1 (5) : 4- 10; Fritze et al. (2006) Biochimica et Biophys Acta. 11 '58: 1633-
1640). In general, the remote loading method involves adding a cargo-trapping reagent {e.g., protonating reagent such as TEA8SOS, ammonium sulfate, etc.) which can be added to the lipid biofilm prior to the sonication in the formation of silicasomes, or can be incorporated into the nanovesicle lipids prior to the formation of the nanovesicle e.g., as described in Example 2.
[0655] Thus for example, as described in Example 2, using the IND-PL prodrug, a DOX/IND nanovesicle cam be prepared as follows: 1) a total of 50 mg lipids of IND-PL plus other vesicle-forming lipids (e.g., IM)-PL/cholesterol/DSPE-PEG2K), in certain
embodiments at a molar ratio, 75:20:5 can be dissolved in 5 mL chloroform in a 50 mL round bottom glass flask. The solvent can be evaporated under a rotatory vacuum to form a uniform thin lipid film, can be dried further under vacuum overnight. 2) The film can be hydrated with a cargo-trapping agent (e.g., with 2 mL of ammonium sulfate (123 mM) and probe sonicated, e.g., for 1 h, then subsequently extruded, e.g., 15 times, through a Mini- Extruder (Avanti Polar Lipids), using, e.g., a polycarbonate membrane with 100 nm pores (Avanti Polar Lipids) at 80 °C. IND nanovesicle (IND-NV) size and morphology can be assessed by dynamic light scattering and cryoEM, respectively as desired. Unincorporated cargo-trapping agent (e.g., ammonium sulfate) can be removed, e.g., by running through a PD-10 size exclusion column. The drug to be loaded (e.g., 6.4 mg of DOX'HCl (10 mg/mL) in DI water) can be incubated with the above prepared IND-NVs, e.g., at 65 °C for 40 min. The nanovesicles can be fractionated across a PD-10 column, allowing the removal of free DOX. Their size and morphology can be assessed by dynamic light scattering, cryoEM and UPLC/MS-MS, respectively.
[0656] Of course, this protocol is illustrative and non-limiting. Using this teaching, numerous other nanovesicles comprising an ICD-inducer and various lipid formulatiosn can be produced by one of skill in the art.
[0657] Similarly, preparation and remote-loading of a silicasome comprising an IDO pathway inhibitor and an ICD-inducer is illustrated in example 2. Figure 6 illustrates the synthesis of DOX-laden IND-PL coated MSNP. A DOX/IND-MSNP dual-delivery carrier was designed by trapping DOX in the mesoporous interior of a -65 nm MSNP, using the lipid bilayer into which IND-PL was incorporated. In order to apply the lipid coating, we used the previously described biofilm method for rapid encapsulation, by sonication (Meng et al. (2015) ACS Nano, 9(4): 540-3557; Liu et al. (2016) ACS Nano, 10: 2702-2715). DOX was then remotely loaded using the protocol as previously described (Id). [0658] Typically this involves preparing the MSNPs, e.g., by a sol-gel synthesis process (see. e.g., Meng et al. (2015) ACS Nano, 9(4): 540-3557). The MSNPs are then soaked in the cargo-trapping agent {e.g., ammonium sulfate) to load the agent into the pores of the MSNPs. The lipid formulation that will comprise the bilayer surrounding the silicasome is prepared, e.g., as described in Example 2, where the lipid formulation incorporates the IDO inhibitor {e.g., IND-PL). The cargo-trapping agent loaded MSNPs are added to the IDO-inhibitor lipid film followed by sonication {e.g., 30 min probe sonication) to provide the trapping agent {e.g., ammonium sulfate)-loaded IND-PL coated MSNP. To remove the free ammonium sulfate, the particle suspension was passed through a PD-10 size exclusion column. Ammonium sulfate-containing IND-PL coated MSNPs were eluted from column faster than free ammonium sulfate due to its large size. Remote Dox loading was accomplished by incubating 6.5-32.4 mg of DOX'HCl (10 mg/mL) in DI water with cargo- trapping agent loaded laden IND-PL coated MSNP at 65 °C for 40 min. The pure DOX/IND- MSNP was collected by centrifuging at 15,000 rpm for 15 min, three times. [0659] This protocol also is illustrative and non-limiting. Using this teaching, numerous other silicasomes comprising an IDO pathway inhibitor and ICD-inducer and various lipid formulatiosn can be produced by one of skill in the art.
[0660] In this regard, it is noted that the lipid conjugation technology described herein can be used to make prodrugs out of chemo agents, which can be folded into a liposome. Thus, for example, ICD chemo agents like the taxanes can be incorporated into a
phospholipid bilayer based on hydrophobicity, and this has been demonstrated for a MSNP where we used paclitaxel incorporation into the encapsulating phospholipid bilayer. The same can be done for a liposome.
[0661] Thus, the versatility of the liposomal platform described herein allows the encapsulation of ICD-inducing drugs such as paclitaxel, docetaxel, mitroxantrone and etoposide through the use different loading strategies that depend on the chemical structure of the drugs. For example, mitoxantrone, which is a weak basic molecule with MW of 444.4, water solubility of 89 mg/mL and log P value of -3.1 (Mitoxantrone.
www.drugbank.ca/drugs/DB01204) , can be remotely loaded into the IND-PL liposome via a proton gradient, using (NH4)2S04. The same is possible for etoposide. Since docetaxel has high ethanol solubility (-100 mg/mL), this lends itself to constructing liposomes by an ethanol injection method that can produce homogeneous unilamellar liposomes as described able. In this method, water is poured into a concentrated lipid-ethanol solution (containing docetaxel and possibly IND-PL in a ratiometric designed strategy), following which ethanol is removed in an evaporator {see, e.g., Pereira et al. (2016) Int. J. Pharmaceutics, , 514: 150- 159). Dilution with water causes spontaneous formation of small and homogenous unilamellar liposomes from the micellar aggregate. The size of the liposomes can be controlled by the ratio of ethanol to water. While paclitaxel (PTX) is moderately soluble in ethanol (1.5 mg/mL), up to ~5 wt% PTX can be loaded into the liposomal membrane by ethanol injection (Koudelka & Turanek(2012) J. Control. Release, 163 : 322-334).
[0662] These emboidments are illustrative and non-limiting. Using the teachings provided herein numerous variants will be available to one of skill in the art. Cargo trapping reagents.
[0663] As explained above, in certain embodiments a cargo-trapping reagent can be utilized to facilitate incorporation of a cargo {e.g., DOX) into the dual-delivery (ICD- inducer/IDO-inhibitor) LB coated MS P (ICD/IDO silicasome), and/or the dual-delivery lipid vesicles {e.g., ICD/IDO-lipid vesicles). The cargo-trapping reagent can be selected to interact with a desired cargo. In some embodiments, this interaction can be an ionic or protonation reaction, although other modes of interaction are contemplated. The cargo- trapping agent can have one or more ionic sites, i.e., can be mono-ionic or poly-ionic. The ionic moiety can be cationic, anionic, or in some cases, the cargo-trapping agent can include both cationic and anionic moieties. The ionic sites can be in equilibrium with corresponding uncharged forms; for example, an anionic carboxylate (-COO") can be in equilibrium with its corresponding carboxylic acid (-COOH); or in another example, an amine (-NH2) can be in equilibrium with its corresponding protonated ammonium form (- H3+). These equilibriums are influenced by the pH of the local environment. Certain ICD-inducing weak-base reagents, such as doxorubicin, can be loaded using a trapping agent mediated approach for loading {see, e.g., Example 2).
[0664] Likewise, in certain embodiments, the cargo can include one or more ionic sites. The cargo-trapping agent and cargo can be selected to interact inside the dual-delivery (ICD-inducer/IDO-inhibitor) LB coated MSNP (ICD/IDO silicasome), and/or the dual- delivery lipid vesicle {e.g., ICD/IDO-lipid vesicle). This interaction can help retain the cargo within the nanoparticle until release of the cargo is desired. In some embodiments, the cargo can exist in a pH-dependent equilibrium between non-ionic and ionic forms. The non-ionic form can diffuse across the lipid bilayer and enter the vesicle or the pores of the MSNP. There, the cargo-trapping agent (e.g., a polyionic cargo-trapping agent) can interact with the ionic form of the cargo and thereby retain the cargo within the nanocarrier, e.g., within the vesicle or within the pores of the MSNP (provided the ionic forms of the cargo and cargo- trapping agent have opposite charges). The interaction can be an ionic interaction, and can include formation of a precipitate. Trapping of cargo within the nanocarrier can provide higher levels of cargo loading compared to similar systems, e.g., nanocarriers that omit the cargo-trapping agent, or liposomes that do include a trapping agent. Release of the cargo can be achieved by an appropriate change in pH to disrupt the interaction between the cargo and cargo-trapping agent, for example, by returning the cargo to its non-ionic state which can more readily diffuse across the lipid bilayer. In one embodiment, the cargo is irinotecan and the cargo-trapping agent is TEA8SOS.
[0665] The cargo trapping agent need not be limited to TEA8SOS. In certain embodiments the cargo trapping comprises small molecules like (NH4)2S04, and the like (see, e.g., Example 2). Other trapping agents include, but are not limited to, ammonium salts (e.g., ammonium sulfate, ammonium sucrose octasulfate, ammonium a-cyclodextrin sulfate, ammonium β-cyclodextrin sulfate, ammonium γ-cyclodextrin sulfate, ammonium phosphate, ammonium a-cyclodextrin phosphate, ammonium β-cyclodextrin phosphate, ammonium γ- cyclodextrin phosphate, ammonium citrate, ammonium acetate, and the like),
trimethylammonium salts (e.g., trimethylammonium sulfate, trimethylammonium sucrose octasulfate, trimethylammonium α-cyclodextrin sulfate, trimethylammonium β-cyclodextrin sulfate, trimethylammonium γ-cyclodextrin sulfate, trimethylammonium phosphate, trimethylammonium α-cyclodextrin phosphate, trimethylammonium β-cyclodextrin phosphate, trimethylammonium γ-cyclodextrin phosphate, trimethylammonium citrate, trimethylammonium acetate, and the like), triethylammonium salts (e.g., triethylammonium sulfate, triethylammonium sucrose octasulfate, triethylammonium a-cyclodextrin sulfate, triethylammonium β-cyclodextrin sulfate, triethylammonium γ-cyclodextrin sulfate, triethylammonium phosphate, triethylammonium α-cyclodextrin phosphate,
triethylammonium β-cyclodextrin phosphate, triethylammonium γ-cyclodextrin phosphate, triethylammonium citrate, triethylammonium acetate, and the like). [0666] It is also worth pointing out that, in addition to TEA8SOS, transmembrane pH gradients can also be generated by acidic buffers (e.g. citrate) (Chou et al. (2003) J. Biosci. Bioengineer., 95(4): 405-408; Nichols et al. (1976) Biochimica et Biophysica Acta (BBA)- Biomembranes, 455(1): 269-271), proton-generating dissociable salts (e.g. (NH4)2S04) (Haran et a/. (1993) Biochimica et Biophysica Acta (BBA)-Biomembranes, 1151(2): 201-215; Maurer-Spurej et al. (1999) Biochimica et Biophysica Acta (BBA)-Biomembranes, 1416(1): 1-10; Fritze et al. (2006) Biochimica et Biophysica Acta (BBA)-Biomembranes, 1758(10): 1633-1640), or ionophore-mediated ion gradients from metal salts (e.g. A23187 and MnS04) (Messerer et al. (2004) Clinical Cancer Res. 10(19): 6638-6649; Ramsay et al. (2008) Eur. J. Pharmaceut. Biopharmaceut. 68(3): 607-617; Fenske et al. (1998) Biochimica et Biophysica Acta (BBA)-Biomembranes, 1414(1): 188-204). Moreover, it is possible to generate reverse pH gradients for drug loading, such as use a calcium acetate gradient to improve amphiphilic weak acid loading in LB-MS P, a strategy that has been utilized in liposomes (Avnir et al. (2008) Arthritis & Rheumatism, 58(1): 119-129).
[0667] In certain embodiments the cargo-trapping reagent is particular suitable for use with a cargo that comprises an organic compound that includes at least one primary amine group, or at least one secondary amine group, or at least one tertiary amine group, or at least one quaternary amine group, or any combination thereof, capable of being protonated. [0668] In certain embodiments the general characteristics of these cargo molecules include the following chemical properties:
[0669] (i) organic molecular compounds that include primary, secondary, tertiary or quaternary amine(s);
[0670] (ii) a pKa <11 to allow protonation and entrapment behind the LB (Zucker et al. (2009) J. Control. Release, 139(1): 73-80; Cern et al. (2012) J. Control.
Release, 160(2): 147-157; Xu et al. (2014) Pharmaceut. Res. 31(10): 2583-2592);
[0671] (iii) a water solubility index of 5-25 mg/mL and amphipathic characteristics that allow diffusion across the LB;
[0672] (iv) an octanol/water partition coefficient or logP value of -3.0 to 3.0 (Zucker et al. (2009) J. Control. Release, 139(1): 73-80; Cern et al. (2012) J. Control.
Release, 160(2): 147-157);
[0673] (v) suitable molecular weight with a geometric size less than MS P pore size (2-8 nm), to allow entry into the MSNP pores (Li et al. (2012) Chem. Soc. Rev. 41(7): 2590-2605; Tang et al. (2012) Adv. Mat. 24(12): 1504-1534; Tarn et al. (2013) ^cc. Chem. Res. 46(3): 792-801).
[0674] Remote loading utilizing doxorubicin, with ammonium sulfate as a cargo trapping agent is described in Example 2. This is illustrative, but non-limiting. In addition to DOX loading into nanovesicles or silicasomes, there are other possible drugs that can be imported across the lipid bilayer of these carriers. These include, but are not limited to, weak basic compounds, with medicinal chemical features. Such compounds include, but are not limited to alkaloids (e.g. irinotecan, topotecan, 10-hydroxycamptothecin, belotecan, rubitecan, vinorelbine, LAQ824, vinblastine, vincristine, homoharringtonine, trabectedin),
anthracyclines (e.g. doxorubicin, epirubicin, pirarubicin, daunorubicin, rubidomycin, valrubicin, amrubicin), alkaline anthracenediones (e.g. mitoxantrone), alkaline alkylating agents (e.g. cyclophosphamide, mechlorethamine, temozolomide), purine or pyrimidine derivatives (e.g. 5-fluorouracil, 5'-deoxy-5-fluorouridine, gemcitabine, capecitabine) and protein kinase inhibitors (e.g., pazopanib, enzastaurin, vandetanib erlotinib, dasatinib, nilotinib, sunitinib, osimertinib, palbociclib, ribociclib), and the like.
[0675] Using the teachings provided herein, numerous other agents can be remote loaded (e.g., loaded using a cargo trapping agent) into the silicasomes (e.g., dual-delivery (ICD-inducer/IDO-inhibitor) LB coated MSNP (ICD/IDO silicasome)), and vesicles (e.g., the dual-delivery lipid vesicles (e.g., ICD/IDO-lipid vesicles)) described herein. Targeting ligands and Immunoconjugates.
[0676] In certain embodiments the dual-delivery (ICD-inducer/IDO-inhibitor) LB coated MSNPs (ICD/IDO silicasomes), and/or the dual-delivery lipid vesicles (e.g.,
ICD/IDO-lipid vesicles), and/or dual delivery lipid-coated ICD-inducing nanomaterial carriers can be conjugated to one or more targeting ligands, e.g., to facilitate specific delivery in endothelial cells, to cancer cells, to fusogenic ligands, e.g., to facilitate endosomal escape, ligands to promote transport across the blood-brain barrier, and the like.
[0677] In one illustrative, but non-limiting embodiment, the nanocarrier (e.g.,
ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) is conjugated to a fusogenic peptide such as histidine-rich H5WYG (H2N- GLFHAIAHFIHGGWHGLIHGWYG-COOH, (SEQ ID NO : 1 )) (see, e.g. , Midoux et al. , (1998) Bioconjug. Chem. 9: 260-267).
[0678] In certain embodiments the nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) is conjugated to one or more targeting ligand(s) that can include antibodies as well as targeting peptides. Targeting antibodies include, but are not limited to intact immunoglobulins, immunoglobulin fragments (e.g., F(ab)'2; Fab, etc.) single chain antibodies, diabodies, affibodies, unibodies, nanobodies, and the like. In certain embodiments antibodies will be used that specifically bind a cancer marker (e.g., a tumor associated antigen). A wide variety of cancer markers are known to those of skill in the art. The markers need not be unique to cancer cells, but can also be effective where the expression of the marker is elevated in a cancer cell (as compared to normal healthy cells) or where the marker is not present at comparable levels in surrounding tissues (especially where the chimeric moiety is delivered locally).
[0679] Illustrative cancer markers include, for example, the tumor marker recognized by the D4 monoclonal antibody. This marker is found on poorly differentiated colorectal cancer, as well as gastrointestinal neuroendocrine tumors (see, e.g., Tobi et al. (1998) Cancer Detection and Prevention, 22(2): 147-152). Other important targets for cancer
immunotherapy are membrane bound complement regulatory glycoproteins CD46, CD55 and CD59, which have been found to be expressed on most tumor cells in vivo and in vitro.
Human mucins (e.g. MUCl) are known tumor markers as are gplOO, tyrosinase, and MAGE, which are found in melanoma. Wild-type Wilms' tumor gene WTl is expressed at high levels not only in most of acute myelocytic, acute lymphocytic, and chronic myelocytic leukemia, but also in various types of solid tumors including lung cancer.
[0680] Acute lymphocytic leukemia has been characterized by the TAAs HLA-Dr,
CD1, CD2, CD5, CD7, CD 19, and CD20. Acute myelogenous leukemia has been
characterized by the TAAs HLA-Dr, CD7, CD13, CD14, CD15, CD33, and CD34. Breast cancer has been characterized by the markers EGFR, HER2, MUCl, Tag-72. Various carcinomas have been characterized by the markers MUCl, TAG-72, and CEA. Chronic lymphocytic leukemia has been characterized by the markers CD3, CD 19, CD20, CD21, CD25, and HLA-DR. Hairy cell leukemia has been characterized by the markers CD 19, CD20, CD21, CD25. Hodgkin's disease has been characterized by the Leu-Ml marker.
Various melanomas have been characterized by the HMB 45 marker. Non-hodgkins lymphomas have been characterized by the CD20, CD 19, and la marker. And various prostate cancers have been characterized by the PSMA and SE10 markers.
[0681] In addition, many kinds of tumor cells display unusual antigens that are either inappropriate for the cell type and/or its environment, or are only normally present during the organisms' development (e.g., fetal antigens). Examples of such antigens include the glycosphingolipid GD2, a disialoganglioside that is normally only expressed at a significant level on the outer surface membranes of neuronal cells, where its exposure to the immune system is limited by the blood-brain barrier. GD2 is expressed on the surfaces of a wide range of tumor cells including neuroblastoma, medulloblastomas, astrocytomas, melanomas, small-cell lung cancer, osteosarcomas and other soft tissue sarcomas. GD2 is thus a convenient tumor-specific target for immunotherapies.
[0682] Other kinds of tumor cells display cell surface receptors that are rare or absent on the surfaces of healthy cells, and which are responsible for activating cellular signaling pathways that cause the unregulated growth and division of the tumor cell. Examples include (ErbB2) HER2/neu, a constitutively active cell surface receptor that is produced at abnormally high levels on the surface of breast cancer tumor cells.
[0683] Other useful targets include, but are not limited to CD20, CD52, CD33, epidermal growth factor receptor and the like. [0684] An illustrative, but not limiting list of suitable tumor markers is provided in
Table 4. Antibodies to these and other cancer markers are known to those of skill in the art and can be obtained commercially or readily produced, e.g. using phage-display technology. Such antibodies can readily be conjugated to the drug delivery nanocarrier (e.g., LB-coated nanoparticle) described herein, e.g., in the same manner that iRGD peptide is conjugated in Example 3.
[0685] Table 4. Illustrative cancer markers and associated references, all of which are incorporated herein by reference for the purpose of identifying the referenced tumor markers.
Figure imgf000127_0001
CD33 Nakase et al. (1996) Am J Clin Pathol, 105(6): 761-768
CD35 Yamakawa et al. Cancer, 73(11): 2808-2817
CD44 Naot et al. (1997) Adv Cancer Res., 71 : 241-319
CD45 Buzzi et al. (1992) Cancer Res., 52(14): 4027-4035
CD46 Yamakawa et al. (1994) Cancer, 73(11): 2808-2817
CD 5 Stein et al. (1991) Clin Exp Immunol, 85(3): 418-423
CD52 Ginaldi et al. (1998) LeukRes., 22(2): 185-191
CD55 Spendlove et al. (1999) Cancer Res., 59: 2282-2286.
CD59 (791Tgp72) Jarvis et a/. (1997) Int J Cancer, 71(6): 1049-1055
CDC27 Wang et al. (1999) Science, 284(5418): 1351-1354
CDK4 Wolfel et al. (1995) Science, 269(5228): 1281-1284
CEA Kass et al. (1999) Cancer Res., 59(3): 676-683
c-myc Watson et al. (1991) Cancer Res., 51(15): 3996-4000
Cox-2 Tsujii et al. (1998) Cell, 93 : 705-716
DCC Gotley et a/. (1996) Oncogene, 13(4): 787-795
DcR3 Pitti et al. (1998) Nature, 396: 699-703
E6/E7 Steller et a/. (1996) Cancer Res., 56(21): 5087-5091
EGFR Yang et a/. (1999) Cancer Res., 59(6): 1236-1243.
EM BP Shiina et a/. (1996) Prostate, 29(3): 169-176.
Ena78 Arenberg et a/. (1998) J. Clin. Invest., 102: 465-472.
FGF8b and FGF8a Dorkin et al. (1999) Oncogene, 18(17): 2755-2761
FLK-l/KDR Annie and Fong (1999) Cancer Res., 59: 99-106
Folic Acid Receptor Dixon et a/. (1992) J Biol Chem., 267(33): 24140-72414
G250 Divgi et al. (1998) C7/w Cancer Res., 4(11): 2729-2739
GAGE-Family De Backer et al. (1999) Cancer Res., 59(13): 3157-3165 gastrin 17 Watson et al. (1995) Int J Cancer, 61(2): 233-240
Gastrin-releasing Wang et al. (1996) Int J Cancer, 68(4): 528-534
hormone (bombesin)
GD2/GD3/GM2 Wiesner and Sweeley (1995) Int J Cancer, 60(3): 294-299
GnRH Bahk et al. (1998) Urol Res., 26(4): 259-264
GnTV Hengstler et a/. (1998) Recent Results Cancer Res., 154: 47-85 gpl00/Pmel l7 Wagner et a/. (1997) Cancer Immunol Immunother., 44(4): 239- 247
gp-100-in4 Kirkin et al. (1998) APMIS, 106(7): 665-679
gpl5 Maeurer et al. (1996) Melanoma Res., 6(1): 11-24
gp75/TRP-l Lewis et a/.(1995) Semin Cancer Biol, 6(6): 321-327 hCG Hoermann et al. (1992) Cancer Res., 52(6): 1520-1524
Heparanase Vlodavsky et al. (1999) Nat Med., 5(7): 793-802 Her2/neu Lewis et al. (1995) Semin Cancer Biol., 6(6): 321-327
Her3
HMTV Kahl et a/.(1991) Br J Cancer, 63(4): 534-540
Hsp70 Jaattela et aZ (199%) EMBO J., 17(21): 6124-6134
hTERT Vonderheide et al. (1999) Immunity, 10: 673-679. 1999.
(telomerase)
IGFR1 Ellis et al. (1998) Breast Cancer Res. Treat., 52: 175-184
IL-13R Murata et al. (1997) Biochem Biophys Res Commun., 238(1): 90-94 iNOS Klotz et aZ (1998) Cancer, 82(10): 1897-1903
Ki 67 Gerdes et aZ (1983) Int J Cancer, 31 : 13-20
KIAA0205 Gueguen et aZ (1998) J Immunol, 160(12): 6188-6194
K-ras, H-ras, Abrams et al. (1996) Semin Oncol, 23(1): 118-134
N-ras
KSA Zhang et al. (1998) Clin Cancer Res., 4(2): 295-302
(CO 17-1 A)
LDLR-FUT Caruso et al. (1998) Oncol Rep., 5(4): 927-930
MAGE Family Marchand et al. (1999) Int J Cancer, 80(2): 219-230
(MAGE1,
MAGE3, etc.)
Mammaglobin Watson et al. (1999) Cancer Res., 59: 13 3028-3031
MAP 17 Kocher et aZ (1996) Am J Pathol, 149(2): 493-500
Melan-A/ Lewis and Houghton (1995) Semin Cancer Biol, 6(6): 321-327 MART-1
mesothelin Chang et al. (1996) Proc. Natl. Acad. Sci., USA, 93(1): 136-140
MIC A/B Groh et a/.(1998) Science, 279: 1737-1740
MT-MMP's, such as Sato and Seiki (1996) J Biochem (Tokyo), 119(2): 209-215 MMP2, MMP3,
MMP7, MMP9
Moxl Candia et aZ (1992) Development, 116(4): 1123-1136
Mucin, such as MUC- Lewis and Houghton (1995) Semin Cancer Biol, 6(6): 321-327 1, MUC-2, MUC-3,
and MUC-4
MUM-1 Kirkin et al. (1998) APMIS, 106(7): 665-679
NY-ESO-1 Jager et al. (1998) J. Exp. Med, 187: 265-270
Osteonectin Graham et al. (1997) Eur J Cancer, 33(10): 1654-1660
pl5 Yoshida et aZ (1995) Cancer Res., 55(13): 2756-2760
P170/MDR1 Trock et al. (1997) J Natl Cancer Inst., 89(13): 917-931 p53 Roth et al. (1996) Proc. Natl. Acad. Sci., USA, 93(10): 4781-4786. p97/melanotransferrin Furukawa et aZ (1989) J Exp Med., 169(2): 585-590
PAI-1 Grandahl-Hansen et aZ (1993) Cancer Res., 53(11): 2513-2521 PDGF Vassbotn et a/. (1993) Mol Cell Biol, 13(7): 4066-4076
Plasminogen (uPA) Naitoh et al. (1995) Jpn J Cancer Res., 86(1): 48-56
PRAME Kirkin et a/. (1998) APMIS, 106(7): 665-679
Probasin Matuo et al. (1985) Biochem Biophys Res Commun., 130(1): 293- 300
Progenipoietin
PSA Sanda et al. (1999) Urology, 53(2): 260-266.
PSM Kawakami et al.(\997) Cancer Res., 57(12): 2321-2324
RAGE-1 Gaugler et a/.(1996) Immunogenetics, 44(5): 323-330
Rb Dosaka-Akita et al. (1997) Cancer, 79(7): 1329-1337
RCAS1 Sonoda et al. (1996) Cancer, 77(8): 1501-1509.
SART-1 Kikuchi et al.(\999( Int J Cancer, 81(3): 459-466
SSX gene Gure et al. (1997) Int J Cancer, 72(6): 965-971
Family
STAT3 Bromberg et al. (1999) Cell, 98(3): 295-303
STn Sandmaier et al. (1999) JImmunother., 22(1): 54-66
(mucin assoc.)
TAG-72 Kuroki et a/. (\990)Cancer Res., 50(16): 4872-4879
TGF-a Imanishi et al. (1989) Br J Cancer, 59(5): 761-765
TGF-β Picon et al. (1998) Cancer Epidemiol Biomarkers Prey, 7(6): 497- 504
Thymosin β 15 Bao et a/. (1996) Nature Medicine . 2(12), 1322-1328
IFN-a Moradi et al. (1993) Cancer, 72(8): 2433-2440
TPA Maulard et al. (1994) Cancer, 73(2): 394-398
TPI Nishida et a/.(1984) Cancer Res 44(8): 3324-9
TRP-2 Parkhurst et al. (1998) Cancer Res., 58(21) 4895-4901
Tyrosinase Kirkin et a/. (1998) APMIS, 106(7): 665-679
VEGF Hyodo et a/. (1998) Eur J Cancer, 34(13): 2041-2045
ZAG Sanchez et al. (1999) Science, 283(5409): 1914-1919
pl6INK4 Quelle et al. (1995) Oncogene Aug. 17, 1995; 11(4): 635-645
Glutathione Hengstler (1998) et al. Recent Results Cancer Res., 154: 47-85
S-transferase
[0686] Any of the foregoing markers can be used as targets for the targeting moieties comprising the nanocarrier (e.g., ICD/IDO silicasomes, ICD/IDO lipid vesicles, ICD- inducing nanomaterial carriers, etc.) constructs described herein. In certain embodiments the target markers include, but are not limited to members of the epidermal growth factor family (e.g., HER2, HER3, EGF, HER4), CDl, CD2, CD3, CD5, CD7, CD13, CD14, CD15, CD19, CD20, CD21, CD23, CD25, CD33, CD34, CD38, 5E10, CEA, HLA-DR, HM 1.24, HMB 45, la, Leu-Ml, MUC1, PMSA, TAG-72, phosphatidyl serine antigen, and the like.
[0687] The foregoing markers are intended to be illustrative and not limiting. Other tumor associated antigens will be known to those of skill in the art. [0688] Where the tumor marker is a cell surface receptor, a ligand to that receptor can function as targeting moieties. Similarly, mimetics of such ligands can also be used as targeting moieties. Thus, in certain embodiments peptide ligands can be used in addition to or in place of various antibodies. An illustrative, but non-limiting list of suitable targeting peptides is shown in Table 5. In certain embodiments any one or more of these peptides can be conjugated to a drug delivery vehicle described herein.
Table 5. Illustrative, but non-limiting peptides that target membrane receptors expressed or overexpressed by various cancer cells.
Figure imgf000131_0001
[0689] In certain embodiments the nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) can be conjugated to moieties that facilitate stability in circulation and/or that hide the nanocarrier from the reticuloendothelial system (REC) and/or that facilitate transport across a barrier (e.g., a stromal barrier, the blood brain barrier, etc.), and/or into a tissue. In certain embodiments the nanocarriers are conjugated to transferrin or ApoE to facilitate transport across the blood brain barrier. In certain embodiments the nanocarriers are conjugated to folate.
[0690] Methods of coupling the nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomatenal carrier, etc.) to targeting (or other) agents are well known to those of skill in the art. Examples include, but are not limited to the use of biotin and avidin or streptavidin (see, e.g., U.S. Patent No: US 4,885, 172 A), by traditional chemical reactions using, for example, bifunctional coupling agents such as glutaraldehyde, diimide esters, aromatic and aliphatic diisocyanates, bis-p-nitrophenyl esters of dicarboxylic acids, aromatic disulfonyl chlorides and bifunctional arylhalides such as l,5-difluoro-2,4- dinitrobenzene; ρ,ρ'-difluoro m,m'-dinitrodiphenyl sulfone, sulfhydryl-reactive maleimides, and the like. Appropriate reactions which may be applied to such couplings are described in Williams et al. Methods in Immunology and Immunochemistry Vol. 1, Academic Press, New York 1967. In one illustrative but non-limiting approach a peptide (e.g., iRGD) is coupled to the (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) by substituting a lipid (e.g., DSPE-PEG2000) with a lipid coupled to a linker (e.g., DSPE- PEG2ooo-maleimide), allowing thiol-maleimide coupling to the cysteine-modified peptide. It will also be recognized that in certain embodiments the targeting (and other) moieties can be conjugated to a other moieties comprising the lipid bilayer on a silicasome or vesicle, or comprising the nanomaterial canier. It is also possible to improve tumor delivery of the IDO inhibitor-ICD inducing nanoparticle, (e.g., OX laden IND-PL-MSNP), through coadministration (not conjugated) of the iRGD peptide to enhance particle transcytosis.
[0691] The former conjugates and coupling methods are illustrative and non-limiting.
Using the teachings provided herein, numerous other moieties can be conjugated to the (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein by any of a variety of methods.
Pharmaceutical Formulations, Administration and Therapy
Pharmaceutical formulations.
[0692] In some embodiments, the nanoca ier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) and/or the ICD-inducing nanomaterials are administered alone or in a mixture with a physiologically-acceptable carrier (such as physiological saline or phosphate buffer) selected in accordance with the route of
administration and standard pharmaceutical practice. For example, when used as an injectable, the nanocarriers can be formulated as a sterile suspension, dispersion, or emulsion with a pharmaceutically acceptable carrier. In certain embodiments normal saline can be employed as the pharmaceutically acceptable carrier. Other suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, 5% glucose and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. In compositions comprising saline or other salt-containing carriers, the carrier is preferably added following nanocarrier formation. Thus, after the nanocarrier is formed and loaded with suitable drug(s), the nanocarrier can be diluted into pharmaceutically acceptable carriers such as normal saline. [0693] Similarly, the ICD-inducing nanomaterials can be introduced into carriers that facilitate suspension of the nanomaterials (e.g., emulsions, dilutions, etc.).
[0694] The pharmaceutical compositions may be sterilized by conventional, well- known sterilization techniques. The resulting aqueous solutions, suspensions, dispersions, emulsions, etc., may be packaged for use or filtered under aseptic conditions. In certain embodiments the drug delivery nanocarriers (e.g., LB-coated nanoparticles) are lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may also contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
[0695] Additionally, in certain embodiments, the pharmaceutical formulation may include lipid-protective agents that protect lipids against free-radical and lipid-peroxidative damage on storage. Lipophilic free-radical quenchers, such as alpha-tocopherol and water- soluble iron-specific chelators, such as ferrioxamine, are suitable. [0696] The concentration of nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) (or ICD-inducing nanomaterial particles) in the pharmaceutical formulations can vary widely, e.g., from less than approximately 0.05%, usually at least approximately 2 to 5% to as much as 10 to 50%, or to 40%, or to 30% by weight and are selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, nanocarriers composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration. The amount of nanocarriers administered will depend upon the particular drug used, the disease state being treated and the judgment of the clinician but will generally be between approximately 0.01 and approximately 50 mg per kilogram of body weight, preferably between approximately 0.1 and approximately 5 mg per kg of body weight.
[0697] In some embodiments, e.g., it is desirable to include polyethylene glycol
(PEG)-modified phospholipids in the LB-coated nanoparticles or vessicles. Alternatively, or additionally, in certain embodiments, PEG-ceramide, or ganglioside GMi-modified lipids can be incorporated in the nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD- inducing nanomaterial carrier, etc.). Addition of such components helps prevent nanocarrier aggregation and provides for increasing circulation lifetime and increasing the delivery of the loaded nanocarriers to the target tissues. In certain embodiments the concentration of the PEG-modified phospholipids, PEG-ceramide, or GMi-modified lipids in the nanocarriers will be approximately 1 to 15%.
[0698] In some embodiments, overall nanocarrier charge is an important determinant in nanocarrier clearance from the blood. It is believed that highly charged nanocarriers (i.e. zeta potential > +35 mV) will be typically taken up more rapidly by the reticuloendothelial system (see, e.g., Juliano (1975) , Biochem. Biophys. Res. Commun. 63 : 651-658 discussing liposome clearance by the RES) and thus have shorter half-lives in the bloodstream.
Nanocarriers with prolonged circulation half-lives are typically desirable for therapeutic uses. For instance, in certain embodiments, drug delivery nanocarriers (e.g., LB-coated
nanoparticles) that are maintained from 8 hrs, or 12 hrs, or 24 hrs, or greater are desirable.
[0699] In another example of their use, nanocarriers (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) can be incorporated into a broad range of topical dosage forms including but not limited to gels, oils, emulsions, and the like, e.g., for the treatment of a topical cancer. For instance, in some embodiments the suspension containing the nanocarrier is formulated and administered as a topical cream, paste, ointment, gel, lotion, and the like. [0700] In some embodiments, pharmaceutical formulations comprising nanocarrier
(e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein additionally incorporate a buffering agent. The buffering agent may be any pharmaceutically acceptable buffering agent. Buffer systems include, but are not limited to citrate buffers, acetate buffers, borate buffers, and phosphate buffers. Examples of buffers include, but are not limited to citric acid, sodium citrate, sodium acetate, acetic acid, sodium phosphate and phosphoric acid, sodium ascorbate, tartaric acid, maleic acid, glycine, sodium lactate, lactic acid, ascorbic acid, imidazole, sodium bicarbonate and carbonic acid, sodium succinate and succinic acid, histidine, and sodium benzoate, benzoic acid, and the like.
[0701] In some embodiments, pharmaceutical formulations comprising nanocarrier
(e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein additionally incorporate a chelating agent. The chelating agent may be any pharmaceutically acceptable chelating agent. Chelating agents include, but are not limited to ethylene diaminetetraacetic acid (also synonymous with EDTA, edetic acid, versene acid, and sequestrene), and EDTA derivatives, such as dipotassium edetate, disodium edetate, edetate calcium disodium, sodium edetate, trisodium edetate, and potassium edetate. Other chelating agents include citric acid (e.g., citric acid monohydrate) and derivatives thereof. Derivatives of citric acid include anhydrous citric acid, tri sodium citrate-dihydrate, and the like. Still other chelating agents include, but are not limited to, niacinamide and derivatives thereof and sodium deoxycholate and derivatives thereof.
[0702] In some embodiments, pharmaceutical formulations comprising nanocarrier
(e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein additionally incorporate an antioxidant. The antioxidant may be any pharmaceutically acceptable antioxidant. Antioxidants are well known to those of ordinary skill in the art and include, but are not limited to, materials such as ascorbic acid, ascorbic acid derivatives (e.g., ascorbylpalmitate, ascorbylstearate, sodium ascorbate, calcium ascorbate, etc.), butylated hydroxy anisole, buylated hydroxy toluene, alkylgallate, sodium meta-bi sulfate, sodium bisulfate, sodium dithionite, sodium thioglycollic acid, sodium formaldehyde sulfoxylate, tocopherol and derivatives thereof, (d-alpha tocopherol, d-alpha tocopherol acetate, dl-alpha tocopherol acetate, d-alpha tocopherol succinate, beta tocopherol, delta tocopherol, gamma tocopherol, and d-alpha tocopherol poly oxy ethylene glycol 1000 succinate) monothioglycerol, sodium sulfite and N-acetyl cysteine. In certain embodiments such materials, when present, are typically added in ranges from 0.01 to 2.0%.
[0703] In some embodiments, pharmaceutical formulations comprising nanocarrier
(e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein are formulated with a cryoprotectant. The cryoprotecting agent may be any pharmaceutically acceptable cryoprotecting agent. Common cryoprotecting agents include, but are not limited to, histidine, polyethylene glycol, polyvinyl pyrrolidine, lactose, sucrose, mannitol, polyols, and the like.
[0704] In some embodiments, pharmaceutical formulations comprising nanocarrier (e-g, ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein are formulated with an isotonic agent. The isotonic agent can be any pharmaceutically acceptable isotonic agent. This term is used in the art interchangeably with iso-osmotic agent, and is known as a compound that is added to the pharmaceutical preparation to increase the osmotic pressure, e.g., in some embodiments to that of 0.9% sodium chloride solution, which is iso-osmotic with human extracellular fluids, such as plasma. Illustrative isotonicity agents include, but are not limited to, sodium chloride, mannitol, sorbitol, lactose, dextrose and glycerol.
[0705] In certain embodiments pharmaceutical formulations of the nanocarrier (e.g.,
ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein may optionally comprise a preservative. Common preservatives include, but are not limited to, those selected from the group consisting of chlorobutanol, parabens, thimerosol, benzyl alcohol, and phenol. Suitable preservatives include but are not limited to: chlorobutanol (e.g., 0.3-0.9% w/v), parabens (e.g., 0.01-5.0%), thimerosal (e.g., 0.004-0.2%), benzyl alcohol (e.g., 0.5-5%), phenol (e.g., 0.1-1.0%), and the like. [0706] In some embodiments, pharmaceutical formulations comprising the nanocarriers (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein are formulated with a humectant, e.g., to provide a pleasant mouth-feel in oral applications. Humectants known in the art include, but are not limited to, cholesterol, fatty acids, glycerin, lauric acid, magnesium stearate, pentaerythritol, and propylene glycol.
[0707] In some embodiments, an emulsifying agent is included in the formulations, for example, to ensure complete dissolution of all excipients, especially hydrophobic components such as benzyl alcohol. Many emulsifiers are known in the art, e.g., polysorbate 60. [0708] For some embodiments related to oral administration, it may be desirable to add a pharmaceutically acceptable flavoring agent and/or sweetener. Compounds such as saccharin, glycerin, simple syrup, and sorbitol are useful as sweeteners. Administration
[0709] The nanocarrier (e.g. , ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD- inducing nanomaterial carrier, etc.) described herein can be administered to a subject (e.g., patient) by any of a variety of techniques. [0710] In certain embodiments the nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) and/or pharmaceutical formulations thereof are administered parenterally, e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the pharmaceutical compositions are administered intravenously, intraarteraly, or intraperitoneally by a bolus injection (see, e.g., U.S. Pat. Nos. 3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578 describing administration of liposomes). Particular pharmaceutical formulations suitable for this administration are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). Typically, the formulations comprise a solution of the drug delivery nanocarrier suspended in an acceptable carrier, preferably an aqueous carrier. As noted above, suitable aqueous solutions include, but are not limited to
physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological (e.g., 0.9% isotonic) saline buffer and/or in certain emulsion formulations. The solution(s) can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In certain embodiments the active agent(s) can be provided in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. For transmucosal
administration, and/or for blood/brain barrier passage, penetrants appropriate to the barrier to be permeated can be used in the formulation. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc., e.g., as described above.
[0711] In other methods, the pharmaceutical formulations containing the nanocarrier
(e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein may be contacted with the target tissue by direct application of the preparation to the tissue. The application may be made by topical, "open" or "closed" procedures. By "topical" it is meant the direct application of the pharmaceutical preparation to a tissue exposed to the environment, such as the skin, oropharynx, external auditory canal, and the like. Open procedures are those procedures that include incising the skin of a patient and directly visualizing the underlying tissue to which the pharmaceutical formulations are applied. This is generally accomplished by a surgical procedure, such as a thoracotomy to access the lungs, abdominal laparotomy to access abdominal viscera, or other direct surgical approaches to the target tissue. Closed procedures are invasive procedures in which the internal target tissues are not directly visualized, but accessed via inserting instruments through small wounds in the skin. For example, the preparations may be administered to the peritoneum by needle lavage. Likewise, the pharmaceutical preparations may be
administered to the meninges or spinal cord by infusion during a lumbar puncture followed by appropriate positioning of the patient as commonly practiced for spinal anesthesia or metrizamide imaging of the spinal cord. Alternatively, the preparations may be administered through endoscopic devices. In certain embodiments the pharmaceutical formulations are introduced via a cannula.
[0712] In certain embodiments the pharmaceutical formulations comprising the nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein are administered via inhalation (e.g., as an aerosol). Inhalation can be a particularly effective delivery route for administration to the lungs and/or to the brain. For administration by inhalation, the drug delivery nanocarriers are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
[0713] In certain embodiments, the nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein are formulated for oral administration. For oral administration, suitable formulations can be readily formulated by combining the drug delivery nanocarriers) with pharmaceutically acceptable carriers suitable for oral delivery well known in the art. Such carriers enable the active agent(s) described herein to be formulated as tablets, pills, dragees, caplets, lozenges, gelcaps, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients can include fillers such as sugars (e.g., lactose, sucrose, mannitol and sorbitol), cellulose preparations (e.g., maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose), synthetic polymers (e.g., polyvinylpyrrolidone (PVP)), granulating agents; and binding agents. If desired, disintegrating agents may be added, such as the cross- linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms may be sugar-coated or enteric-coated using standard techniques. The preparation of enteric-coated particles is disclosed for example in U.S. Pat. Nos.
4,786,505 and 4,853,230.
[0714] In various embodiments the nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described hereien can be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. Methods of formulating active agents for rectal or vaginal delivery are well known to those of skill in the art (see, e.g., Allen (2007) Suppositories, Pharmaceutical Press) and typically involve combining the active agents with a suitable base (e.g., hydrophilic (PEG), lipophilic materials such as cocoa butter or Witepsol W45), amphiphilic materials such as Suppocire AP and polyglycolized glyceride, and the like). The base is selected and compounded for a desired melting/delivery profile.
[0715] The route of delivery of the nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein can also affect their distribution in the body. Passive delivery of nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) involves the use of various routes of administration e.g., parenterally, although other effective administration forms, such as intraarticular injection, inhalant mists, orally active formulations, transdermal iontophoresis, or suppositories are also envisioned. Each route produces differences in localization of the drug delivery nanocarrier.
[0716] Because dosage regimens for pharmaceutical agents are well known to medical practitioners, the amount of the liposomal pharmaceutical agent formulations that is effective or therapeutic for the treatment of a disease or condition in mammals and particularly in humans will be apparent to those skilled in the art. The optimal quantity and spacing of individual dosages of the formulations herein will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and such optima can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, e.g., the number of doses given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
[0717] Typically, the nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) and/or pharmaceutical formations thereof described herein are used therapeutically in animals (including man) in the treatment of various cancers. In certain embodiments the nanocarriers and/or pharmaceutical formations thereof described herein are particularly well suited in conditions that require: (1) repeated administrations; and/or (2) the sustained delivery of the drug in its bioactive form; and/or (3) the decreased toxicity with suitable efficacy compared with the free drug(s) in question. In various embodiments the nanocarriers and/or pharmaceutical formations thereof are administered in a therapeutically effective dose. The term "therapeutically effective" as it pertains to the nanocarriers described herein and formulations thereof means that the combination of ICD inducer and IDO inhibitor produces a desirable effect on the cancer. Such desirable effects include, but are not limited to slowing and/or stopping tumor growth and/or proliferation and/or slowing and/or stopping proliferation of metastatic cells, reduction in size and/or number of tumors, and/or elimination of tumor cells and/or metastatic cells, and/or prevention of recurrence of the cancer following remission.
[0718] Exact dosages will vary depending upon such factors as the particular ICD inducer(s) and IDO inhibitors and the desirable medical effect, as well as patient factors such as age, sex, general condition, and the like. Those of skill in the art can readily take these factors into account and use them to establish effective therapeutic concentrations without resort to undue experimentation.
[0719] For administration to humans (or to non-human mammals) in the curative, remissive, retardive, or prophylactic treatment of diseases the prescribing physician will ultimately determine the appropriate dosage of the drug for a given human (or non-human) subject, and this can be expected to vary according to the age, weight, and response of the individual as well as the nature and severity of the patient's disease. In certain embodiments the dosage of the drug provided by the nanocarrier(s) can be approximately equal to that employed for the free drug. However as noted above, the nanocarriers described herein can significantly reduce the toxicity of the drug(s) administered thereby and significantly increase a therapeutic window. Accordingly, in some cases dosages in excess of those prescribed for the free drug(s) will be utilized.
[0720] In certain embodiments, the dose of each of the drug(s) (e.g., ICD inducer,
IDO inhibitor) administered at a particular time point will be in the range from about 1 to about 1,000 mg/m2/day, or to about 800 mg/m2/day, or to about 600 mg/m2/day, or to about 400 mg/m2/day. For example, in certain embodiments a dosage (dosage regiment) is utilized that provides a range from about 1 to about 350 mg/m2/day, 1 to about 300 mg/m2/day, 1 to about 250 mg/m2/day, 1 to about 200 mg/m2/day, 1 to about 150 mg/m2/day, 1 to about 100 mg/m2/day, from about 5 to about 80 mg/m2/day, from about 5 to about 70 mg/m2/day, from about 5 to about 60 mg/m2/day, from about 5 to about 50 mg/m2/day, from about 5 to about 40 mg/m2/day, from about 5 to about 20 mg/m2/day, from about 10 to about 80 mg/m2/day, from about 10 to about 70 mg/m2/day, from about 10 to about 60 mg/m2/day, from about 10 to about 50 mg/m2/day, from about 10 to about 40 mg/m2/day, from about 10 to about 20 mg/m2/day, from about 20 to about 40 mg/m2/day, from about 20 to about 50 mg/m2/day, from about 20 to about 90 mg/m2/day, from about 30 to about 80 mg/m2/day, from about 40 to about 90 mg/m2/day, from about 40 to about 100 mg/m2/day, from about 80 to about 150 mg/m2/day, from about 80 to about 140 mg/m2/day, from about 80 to about 135 mg/m2/day, from about 80 to about 130 mg/m2/day, from about 80 to about 120 mg/m2/day, from about 85 to about 140 mg/m2/day, from about 85 to about 135 mg/m2/day, from about 85 to about 135 mg/m2/day, from about 85 to about 130 mg/m2/day, or from about 85 to about 120 mg/m2/day. In certain embodiments the does administered at a particular time point may also be about 130 mg/m2/day, about 120 mg/m2/day, about 100 mg/m2/day, about 90 mg/m2/day, about 85 mg/m2/day, about 80 mg/m2/day, about 70 mg/m2/day, about 60 mg/m2/day, about 50 mg/m2/day, about 40 mg/m2/day, about 30 mg/m2/day, about 20 mg/m2/day, about 15 mg/m2/day, or about 10 mg/m2/day.
[0721] Dosages may also be estimated using in vivo animal models, as will be appreciated by those skill in the art. In this regard, with respect to the irinotecan-loaded drug delivery nanocarriers described herein, it is noted that the effective therapeutic dose of the OX/IND nanocarrier in a KPC-derived orthotopic animal model is about 5 mg OX/kg with 50 mg IND/kg, which is equivalent to 15.5 mg OX/m2 IND 150 mg/m2in a 60 kg human subject. Fibonacci analysis indicates this dose can be achieved by starting and intermediary OX doses of 37.5 and 75 mg/m2. It is noted that 75 mg/m2 OX is quite conservative and higher dosages are contemplated.
[0722] The dose administered may be higher or lower than the dose ranges described herein, depending upon, among other factors, the bioavailability of the composition, the tolerance of the individual to adverse side effects, the mode of administration and various factors discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the composition that are sufficient to maintain therapeutic effect, according to the judgment of the prescribing physician. Skilled artisans will be able to optimize effective local dosages without undue experimentation in view of the teaching provided herein.
[0723] Multiple doses (e.g., continuous or bolus) of the compositions as described herein may also be administered to individuals in need thereof of the course of hours, days, weeks, or months. For example, but not limited to, 1, 2, 3, 4, 5, or 6 times daily, every other day, every 10 days, weekly, monthly, twice weekly, three times a week, twice monthly, three times a month, four times a month, five times a month, every other month, every third month, every fourth month, etc.
Methods of treatment.
[0724] In various embodiments methods of treatment using the nanocarrier (e.g. , ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) and/or pharmaceutical formulation(s) comprising nanoparticle drug carriers described herein are provided. In certain embodiments the method(s) comprise a method of treating a cancer. In certain embodiments the method can comprise administering to a subject in need thereof an effective amount of a nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD- inducing nanomaterial carrier, etc.), and/or a pharmaceutical formulation comprising a nanocarrier as described herein, where the drug(s) comprising the nanocarrier and/or said pharmaceutical formulation comprises an anti-cancer drug.
[0725] In certain embodiments the nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) and/or pharmaceutical formulation is a primary therapy in a chemotherapeutic regimen. In certain embodiments the nanoparticle drug carrier and/or pharmaceutical formulation is a component in an adjunct therapy in addition to chemotherapy using one or more other chemotherapeutic agents, and/or surgical resection of a tumor mass, and/or radiotherapy.
[0726] In certain embodiments the nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) and/or pharmaceutical formulation is a component in a multi-drug chemotherapeutic regimen. In certain embodiments the multidrug chemotherapeutic regimen comprises at least two drugs selected from the group consisting of irinotecan (IRIN), oxaliplatin (OX), 5-fluorouracil (5-FU), and leucovorin (LV). In certain embodiments the multi-drug chemotherapeutic regimen comprises at least three drugs selected from the group consisting of irinotecan (IRIN), oxaliplatin (OX), 5- fluorouracil (5-FU), and leucovorin (LV). In certain embodiments the multi-drug
chemotherapeutic regimen comprises at least irinotecan (IRIN), oxaliplatin (OX), 5- fluorouracil (5-FU), and leucovorin (LV).
[0727] In various embodiments nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) and/or pharmaceutical formulation(s) threeof described herein are effective for treating any of a variety of cancers. In certain embodiments the cancer is pancreatic ductal adenocarcinoma (PDAC). In certain
embodiments the cancer is a cancer selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi sarcoma, lymphoma), anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, glioblastoma, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma,
ependymoma, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kaposi sarcoma, kidney cancer (e.g., renal cell, Wilm's tumor, and other kidney tumors), langerhans cell histiocytosis, laryngeal cancer, leukemia, acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic
myelogenous (CML), hairy cell, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., childhood, non-small cell, small cell), lymphoma (e.g., AIDS-related, Burkitt (e.g., non-Hodgkin lymphoma), cutaneous T-Cell (e.g., mycosis fungoides, Sezary syndrome), Hodgkin, non-Hodgkin, primary central nervous system (CNS)), macroglobulinemia, Waldenstrom, male breast cancer, malignant fibrous
histiocytoma of bone and osteosarcoma, melanoma (e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple
myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, chronic myeloid leukemia (CML), multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer , pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Ewing, Kaposi, osteosarcoma, rhadomyosarcoma, soft tissue, uterine), Sezary syndrome, skin cancer (e.g., melanoma, merkel cell carcinoma, basal cell carcinoma, nonmelanoma), small intestine cancer, squamous cell carcinoma, squamous neck cancer with occult primary, stomach (gastric) cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom
macroglobulinemia, and Wilm's tumor. [0728] In certain embodiments the nanocarrier (e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein is not conjugated to an iRGD peptide and the nanocarrier is administered in conjunction with an iRGD peptide (e.g., the nanocarrier and the iRGD peptide are co-administered as separate formulations). [0729] In various embodiments of these treatment methods, the nanocarrier (e.g.,
ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) and/or pharmaceutical formulation is administered via a route selected from the group consisting of intravenous administration, intraarterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation (including intranasal and intratracheal delivery, intracranial administration via a cannula, and subcutaneous or intramuscular depot deposition. In certain embodiments the nanocarrier and/or pharmaceutical formulation is administered as an injection, from an IV drip bag, or via a drug-delivery cannula. In various embodiments the subject is a human and in other embodiments the subject is a non-human mammal. Combined treatment with checkpoint inhibitors.
[0730] It was surprising discovery that the nanocarriers described herein (e.g., comprising an inducer of immunogenic cell death (ICD), and an IDO pathway inhibitor) showed synertistic anti-cancer activity when administered in combination with one or more checkpoint hinibitors. Accordingly, certain embodiments, methods contemplated herein include the administration of a drug delivery nanovesicle and/or a drug delivery nanocarrier as described herein in conjunction with one or more checkpoint inhibitors.
[0731] Illustrative checkpoint inhibitors include, but are not limited to inhibitors of
PD-1, PD-L1, PD-L2, PD-L3, PD-L4, CTLA-4, LAG3, B7-H3, B7-H4, KIR and/or TIM3 receptors. [0732] In some embodiments, the immune checkpoint inhibitor can be a small peptide agent that can inhibit regulatory T cell function, including the inhibitory receptors listed above. In some embodiments, the immune checkpoint inhibitor can be a small molecule (e.g. less than 500 Daltons) that can inhibit T regulatory cell function, including the immune checkpoint receptors listed above. In some embodiments, the immune checkpoint inhibitor can be a molecule providing co-stimulation of T-cell activation. In some embodiments, the immune checkpoint inhibitor can be a molecule providing co-stimulation of natural killer cell activation. In some embodiments, the immune checkpoint inhibitor can be an antibody. In some embodiments, the immune checkpoint inhibitor is a PD-1 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L1 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L2 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L3 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L4 antibody. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antibody. In some embodiments, the immune checkpoint inhibitor is an antibody of CTLA-4, LAG3, B7-H3, B7-H4, KIR, or TIM3.
[0733] In certain embodiments the antibody can be selected from a-CD3-APC, a-
CD3-APC-H7, a-CD4-ECD, a-CD4-PB, a-CD8-PE-Cy7, a-CD-8-PerCP-Cy5.5, a-CD l lc- APC, a-CDl lb-PE-Cy7, a-CDl lb-AF700, a-CD 14-FITC, a-CD16-PB, a-CD19-AF780, a- CD19-AF700, a-CD20-PO, a-CD25-PE-Cy7, a-CD40-APC, a-CD45-Biotin, Streptavidin- BV605, a-CD62L-ECD, a-CD69-APC-Cy7, a-CD80-FITC, a-CD83-Biotin, Streptavidin- PE-Cy7, a-CD86-PE-Cy7, a-CD86-PE, a-CD123-PE, a-CD154-PE, a-CD161-PE, a- CTLA4-PE-Cy7, a-FoxP3-AF488 (clone 259D), IgGl-isotype-AF488, a-ICOS (CD278)-PE, a-HLA-A2-PE, a-HLA-DR-PB, a-HLA-DR-PerCPCy5.5, a-PDl-APC, VISTA, co- stimulatory molecule OX40, CD 137, and the like.
[0734] Any of a variety of antibodies can be used in the methods described herein, including, but nor limited to antibodies having high-affinity binding to PD-1 PD-L1, PD-L2, PD-L3, or PD-L4. Human mAbs (HuMAbs) that bind specifically to PD-1 (e.g., bind to human PD-1 and may cross-react with PD-1 from other species, such as cynomolgus monkey) with high affinity have been disclosed in U.S. Pat. No. 8,008,449, which is incorporated herein by reference for the antibodies described herein. HuMAbs that bind specifically to PD-L1 with high affinity have been disclosed in U.S. Pat. No. 7,943,743, which is incorporated herein by reference for the antibodies described herein. Other anti-PD- 1 mAbs have been described in, for example, U.S. Pat. Nos. 6,808,710, 7,488,802 and
8, 168,757, and PCT Publication No. WO 2012/145493, all of which are incorporated herein by reference for the antibodies described herein. Anti-PD-Ll mAbs have been described in, for example, U.S. Pat. Nos. 7,635,757 and 8,217, 149, U. S. Publication No. 2009/0317368, and PCT Publication Nos. WO 201 1/066389 and WO 2012/14549, all of which are incorporated herein by reference for the antibodies described herein.
[0735] In some embodiments, the anti-PD-1 HuMAbs can be selected from 17D8,
2D3, 4H1, 5C4 (also referred to herein as nivolumab), 4A1 1, 7D3 and 5F4, all of which are described in U.S. Pat. No. 8,008,449. In some embodiments, the anti-PD-1 HuMAbs can be selected from 3G10, 12A4 (also referred to herein as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 1 1E6, 12B7, and 13G4, all of which are described in U.S. Pat. No. 7,943,743.
[0736] In certain embodiments the antibodies comprises antibodies are approved for clinical use. Such antibodies include, but are not limited to antibodies that target PD-1 (e.g., Pembrolizumab (Keytruda), Nivolumab (Opdivo)), antibodies that target PD-L1 (e.g., Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), and the like), and/or antibodies that target CTLA-4 (e.g., Ipilimumab (Yervoy)).
[0737] The foregoing checkpoint inhibitors are illustrative and not limiting. Using the teaching provided herein numerous other checkpoint inhibitors can be used in conjunction with the delivery vehicles described herein.
Kits.
[0738] In certain embodiments, kits are provided containing reagents for the practice of any of the methods described herein. In certain embodiments the kit comprises a container containing an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway (IDO inhibitor); and/or a container containing an agent that induces immunogenic cell death (ICD) (ICD- inducer). In certain embodiments the IDO inhibitor comprises an agent selected from the group consisting of 1-methyl-D-tiyptophan (indoximod), 1-methyl-L-tiyptophan,
methylthiohydantoin-dl -tryptophan, Necrostatin-1, Ebselen, Pyridoxal Isonicotinoyl
Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride,
Norharmane hydrochloride, INCB024360, S-allyl-brassinin, S-benzyl-brassinin, 5-Bromo- brassinin, 4-phenylimidazole Exiguamine A, and NSC401366. In certain embodiments the IDO inhibitor comprises an agent shown in Table 3, supra. In certain embodiments the IDO inhibitor comprises indoximod. In certain embodiments the IDO inhibitor is conjugated to an agent that forms a vesicle. In certain embodiments the agent is selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid. In certain embodiments the agent comprises a phospholipid. In certain embodiments the IDO inhibitor is IDO-PL.
[0739] In certain embodiments the ICD inducer comprises a chemotherapeutic agent selected from the group consisting of oxaliplatin, cisplatin, doxorubicin, epirubicin, idarubicin, mitoxantrone, anthracenedione, bleomycin, bortezomib, R2016, and
cyclophosphamide. In certain embodiments the ICD inducer comprises oxaliplatin. In certain embodiments the ICD inducer is a compound or a biological agent in Table 2. [0740] In certain embodiments the kit contains both an IDO inhibitor and an ICD inducer. In certain embodiments the IDO inhibitor and the ICD inducer are in separate containers. In certain embodiments the IDO inhibitor and said ICD inducer are in the same container. In certain embodiments the IDO inhibitor and said ICD inducer are provided as a nanoparticle drug carrier (e.g., a drug delivery nanocarrier) as described herein.
[0741] In certain embodiments the kit contains an ICD inducer that comprise a nanomaterial or a formulation thereof (e.g., a sterile formulation). In certain embodiments the nanomaterial comprises a material selected form the group consisting of CuO, Sb203, ZnO, Ti02, and graphene oxide. [0742] In certain embodiments the kit comprises a container containing a nanocarrier
(e.g., ICD/IDO silicasome, ICD/IDO lipid vesicle, ICD-inducing nanomaterial carrier, etc.) described herein.
[0743] Additionally, in certain embodiments, the kits can include instructional materials disclosing the means of the use of the ICD inducer to induce immunogenic death in cancer cells for vaccination, and/or the use of the ICD inducer and the IDO inhibitor as a cancer therapeutic for local administration, and/or the use of a drug-loaded drug delivery nanocarrier (e.g., LB-coated nanoparticle) or nanocarrier immunoconjugate as a therapeutic for a cancer (e.g., a pancreatic cancer, gastric cancer, cervical cancer, ovarian cancer, etc.).
[0744] In addition, the kits optionally include labeling and/or instructional materials providing directions (e.g., protocols) for the use of the materials described herein, e.g., alone or in combination for the treatment of various cancers. Instructional materials can also include recommended dosages, description(s) of counterindications, and the like.
[0745] While the instructional materials in the various kits typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
EXAMPLES
[0746] The following examples are offered to illustrate, but not to limit the claimed invention. Example 1
IDO Inhibitor Prodrugs
[0747] Indoleamine-2,3-dioxygenase (IDO) is an intracellular heme-containing enzyme that initiates the first and rate-limiting step of tryptophan degradation along the kynurenine pathway. In mammalian organisms, tryptophan is an essential amino acid for cell survival; it cannot be synthesized de novo. IDO was shown to be expressed in normal tissues such as the endothelial cells in the placenta and lung, the epithelial cells in the female genital tract, and the lymphoid tissues in mature dendritic cells. Munn et al. showed that IDO has a central role in preventing T cell-driven rejection of allogeneic fetuses during pregnancy as trophoblast expressing IDO was found to induce maternal tolerance to fetal allograft {see, e.g., Munn et al. (1998) Science, 281(5380): 1191-1193). This discovery broke ground for further research addressing the immunomodulatory potential of IDO, including the discovery of IDO inhibitor for cancer treatment. The immunosuppressive roles of IDO have also been investigated for elucidation of therapeutic targets in the management of many diseases including cancer (Gajewski et al. (2013) Nature Immunol. 14: 1014-1022; Moon et al. (2015) J. ImmunoTherapy Cancer, 3 : 51).
[0748] Based on a summary of clinical trials (Vacchelli et a/.(2014) Oncoimmunology,
3(10): e957994), we found that:
[0749] 1) Use of indoximod as a standalone agent often fails to cause tumor eradication; and
[0750] 2) Combination therapy, i.e. chemo + indoximod, showed promising results. This includes the use of IDO inhibitor plus many standard chemoagents, such as paclitaxel, docetaxel, etc. In PDAC, a clinical trial using IDO inhibitor plus GEM and PTX is ongoing. Synthesis of IDO inhibitor indoximod prodrug
[0751] Indoximod is a potent IDO pathway inhibitor. It is currently used as its free form in NaOH solution and/or pellets in clinical trials. However, in order to achieve an effective therapeutic dose, extremely high concentrations of Indoximod are required to be used {e.g. oral formulation, 1200-2000 mg/day). We propose to use a bio-conjugation or supramolecular assembly approach to further improve the PK/PD, local retention and potency of Indoximod in vivo, either via local intratumoral injection or systemic IV injection. Bio-conjugation approach
[0752] Indoximod has a functional carboxyl group (see, e.g., Figure 2), that can be readily conjugated to other compounds containing a hydroxyl moiety. A few representative compounds are provided (see, e.g., Figure 3). The resulting pro-drugs can form nanovesicles in the aqueous solution at certain concentrations (e.g. >CMC) or be used as a component to coat MSNPs as described herein, leading to a variety of immunotherapy drug delivery nanocarrier(s) (e.g., LB-coated nanoparticle(s)). Since the resulting conjugates are amphipathic molecules, they are readily incorporated into lipid vesicles and can also self- assemble as micellar structures, both of which are pharmaceutically active. [0753] In certain embodiments a little more complicated ester-mediated conjugation could include the use of linkers such as an HO-(CH2)n=2-5-OH as a linker in the reaction. The cases of oleic acid and docosahexaenoic acid (DHA) fall into this category (see, e.g., Figure 4).
Supramolecular approach:
[0754] It is possible to take advantage of the chemical structure of indoximod or other
IDO inhibitors, allowing the supramolecular assembly of this compound onto a
nanostructured surface mediated by individual or combined molecule-nanomaterial interactions, such as pi-pi stacking, electrostatic interactions, van der Waals' force and/or physical absorption. One example is graphene oxide, which is also an inducer of ICD in our HTS studies.
[0755] While the above-identified methods are illustrated with respect to indoximod, it will be recognized that these or similar methods can be utilized with numerous other IDO inhibitors (see, e.g., Table 3, above, and Figure 2).
Example 2
Self-Asssembled Nanovesicles for the Co-Delivery of an IDO Pathway Inhibitor
Prodrug and Remote Loading of an Immunogenic Cell Death Inducing Agent.
[0756] A potential limitation of the OX/IND-MSNP carrier is its relatively low loading capacity for Pt-based drugs, such as OX (i.e. <10% wt). Since Pt-drugs are coordination complex compounds, they are usually not suitable for remote loading by a proton gradient, such as has been reported for irinotecan encapsulation in LB-coated MSNPs {see, e.g., Liu et al. (2016) ACS Na o, 10: 2702-2715). We therefore developed new particle iterations capable of achieving a higher loading capacity for ICD-inducing chemo agents.
Synthesis of a Dox nanovesicle carrier that co-delivers IND-PL.
[0757] DOX was chosen for remote loading of IND-NVs based on its composition as a weak basic substance. Following its import into the vesicles, DOX precipitated as crystals, yielding a carrier that morphologically resembles the DOXIL® liposome. We consider the DOX/IND-NV as a leading carrier prototype for initiating antitumor immunotherapy in settings such as breast cancer and other cancer types.
Synthesis of DOX IND nanovesicle:
[0758] The IND-PL prodrug synthesis is described in Example 2. Using the IND-PL prodrug, a DOX/IND nanovesicle was prepared as follows: 1) a total of 50 mg lipids of IND- PL/chol ester ol/DSPE-PEG2K (molar ratio, 75:20:5) was dissolved in 5 mL chloroform in a 50 mL round bottom glass flask. The solvent was evaporated under a rotatory vacuum to form a uniform thin lipid film, which was dried further under vacuum overnight. 2) The film was hydrated with 2 mL of ammonium sulfate (123 mM) and probe sonicated for 1 h, which was subsequently extruded 15 times through a Mini -Extruder (Avanti Polar Lipids), using a polycarbonate membrane with 100 nm pores (Avanti Polar Lipids) at 80 °C. IND-NV size and morphology were assessed by dynamic light scattering and cryoEM, respectively. 3) Unincorporated ammonium sulfate was removed by running through a PD-10 size exclusion column. 4) 6.4 mg of DOX'HCl (10 mg/mL) in DI water was incubated with the above prepared IND-NVs at 65 °C for 40 min. 5) The nanovesicles were fractionated across a PD- 10 column, allowing the removal of free DOX. Their size and morphology were assessed by dynamic light scattering, cryoEM and UPLC/MS-MS, respectively. The final product (Figure 5B) was stored at 4°C in the dark prior to biological testing. [0759] The DOX/IND nanovesicle was constructed by self-assembly of IND-PL (see, e.g., Figure 5A). The prodrug is amphipathic, allowing self-assembly into nanovesicles (IND-NV) in the presence of an aqueous biological buffer. Moreover, the entrapment of a protonating agent (such as ammonium sulfate) at the time of self-assembly, permits the nanovesicle to import DOX from the surrounding drug suspension. DOX precipitates as crystals in the nanovesicle, as shown in the Cryo-electron microscopy picture in Figure 5B (top). This provides a nanocarrier, which morphologically resembles the DOXIL® liposome (Figure 5B (bottom). Additional possible weak base laden co-delivery IND-NVs,
[0760] In addition to DOX loading into nanovesicles, there are other possible drugs that can be imported across the lipid bilayer of this carrier. These include, but are not limited to weak basic compounds with medicinal chemical features. Such copounds include, but are not limited to alkaloids (e.g. irinotecan, topotecan, 10-hydroxycamptothecin, belotecan, rubitecan, vinorelbine, LAQ824, vinblastine, vincristine, homoharringtonine, trabectedin), anthracyclines (e.g. doxorubicin, epirubicin, pirarubicin, daunorubicin, rubidomycin, valrubicin, amrubicin), alkaline anthracenediones (e.g. mitoxantrone), alkaline alkylating agents (e.g. cyclophosphamide, mechlorethamine, temozolomide), purine or pyrimidine derivatives (e.g. 5-fluorouracil, 5'-deoxy-5-fluorouridine, gemcitabine, capecitabine) and protein kinase inhibitors (e.g., pazopanib, enzastaurin, vandetanib erlotinib, dasatinib, nilotinib, sunitinib, osimertinib, palbociclib, ribociclib), etc.
Cellular and animal studies.
[0761] IDO-mediated Trp depletion and Kyn accumulation leads to the perturbation of downstream effector pathways involved in immune suppression. Key among these, is the mTOR pathway that is sensitive to Tryp depletion, resulting in interference in the
phosphorylation and activation of P-S6 kinase (P-S6K). 4T1 cells were treated for 3 h in tryptophan-deficient medium, following the 24 h incubation in tryptophan-deficient medium with indicated treatment. Western blot assays (Figure 7) showing the enhanced effect of the IND-nanovesicle on mTOR signaling, which can be studied by assessing the phosphorylation of P-S6K. Free drug at identical dose was used for comparison.
[0762] Figs. 8A to 8D show the results of a biodistribution study in 4T1 orthotopic model. 4Tl-luc cells were orthotopically implanted into mammary fat pad of Balb/c mice. Once the primary tumor reaches -200 mm3 in size (a stage without obvious metastasis), IVIS imaging (Figure 8A) was performed to look at primary tumor burden by bioluminescence.
Figure 8B shows images of the same model shown in (Figure 8 A) after receiving IV injection of free Dox, DOXIL® and Dox/IND-NV at identical Dox dose (5mg/kg). The animals were sacrificed 24 hrs post injection. Tumor and major organs were collected for ex vivo imaging. IVIS system was also used to visualize DOX fluorescent intensity in the tumor (yellow circles) and major organs. In a separate PK study, single IV injection of free DOX, DOXIL® or DOX/IND-NV (Dox 5 mg/kg) was carried out. At 0.08333, 0.25, 0.5, 1, 2.5, 8, 24, 48 h post injection, blood was drawn and subject to the DOX and IND concentration determination (Figure 8C). Also, at 24 h, 3 of the 6 mice were sacrificed, and major organs were isolated for the drug content analysis. For DOX/IND-NV treated mice in (Figure 8C), we also measure indoximod concentration using FIPLC (Figure 8D). For ease of data visualization, PK profile of DOX/IND-NV was present in terms of Dox or IND
concentrations.
[0763] Figs 9A and 9B show the results of an ongoing anti-cancer efficacy study in
4T1 orthotopic breast cancer bearing mice. One million 4Tl-luc cells were injected into the mammary fat pad of Balb/c mice at day 0. The treatments were launched when the tumor size reached around -100 mm3 (day 8). A total of 4 IV injections of Dox/IND-NV (Dox 5 mg/kg; IND-PL 31.5 mg/kg; molar ratio of DOX:IND-PL = 1 :5) were performed (indicated by arrow in Figure 9A). Controls include saline, free Dox, DOXIL® liposome and free Dox + IND- NV at identical drug doses. Tumor size was measured 2-3 times per week. While the animal efficacy experiment is still ongoing, a promising anticancer trend already emerges in mice receiving Dox/IND-NV. In fact, in the experiment shown in (Figure 9 A), we also included additional treatment using free DOX plus anti-PDl (Figure 9B) with a view to demonstrate the advantage of DOX/IND-NV versus a standard chemo/immuno combination therapy in breast cancer. Please note that 4T1 tumor that we grew was PD-1+, as confirmed by IHC. Based on the promising efficacy result, it is believed that a statistically significant efficacy improvement is observed when the mice received Dox/IND-NV versus various controls. Example 3
Tailor-design of a co-delivery liposome for breast cancer nano-immunotherapy by contemporaneous triggering of immunogenic cell death and restraining the IDO pathway
[0764] While treatment of patients with localized breast cancer (BC) has a survival rate of -98%, the Breast Cancer Coalition has pointed out that there is marginal improvement on mortality rate since 1975 (DeSantis et al. (2017) CA Cancer J Clin. 67: 439-448). This is particularly true for metastatic disease, where none of the current treatments {e.g., radiation, chemotherapy, and estrogen blockers) are capable of eliminating BC once metastatic spread has taken place (Howlader et al. (eds). SEER Cancer Statistics Review, 1975-2010, Nat. Cancer Inst. Bethesda, MD, seer.cancer.gov/csr/1975_2010/, based on November 2012 SEER data submission, posted to the SEER web site, April 2013). Newfound optimism has emerged with the advent of cancer immunotherapy, where the power of T-cell immunity can be invoked to treat solid cancers, including breast cancer (Em ens (2018) Clin. Cane. Res. 24: 511-520). This is best exemplified by the use of immune checkpoint blocking antibodies, that at have changed the treatment landscape for melanoma and non-small cell lung cancer (NSCLC) {Id.). However, in spite of this accomplishment, the overall response rate is only 20-30%, without clear guidance to identify responders {see, e.g., Solinas et al. (2017) ESMO Open, 2: e000255).
[0765] The overarching challenge that we address to improve BC mortality is to improve the response rate to immunotherapy through the delivery of immunogenic cell death (ICD) stimuli by nanocarriers (Fig. 1). Our data show reproducible induction of tumor infiltrating lymphocytes (TILs) in an orthotopic BC animal model by an ICD-inducing nanocarrier. The advantage of using a nanocarrier to deliver ICD-inducing chemotherapy to the cancer site lies in its improved pharmacokinetics, and decreased toxicity of the drugs. This will eliminate the guesswork to find responders, who are postulated to be patients with a high mutational load, in whom non-synonymous mutations generate a "hot" immune environment (TME) (Nagarsheth et al. (2017) Nat. Rev. Immunol. 17: 559). This facilitates boosting of the immune response by antibodies that block CTLA-4, PD-1 and, PD-L1 receptors.
[0766] We propose that ICD will allow more predictable induction of an immune replete status to allow receptor-mediated blockade or perturbation of other immune surveillance pathways to induce durable anti-tumor immunity, which also takes care of metastases. As such, ICD could strengthen the effect of immune checkpoint blocking antibodies as well as indoleamine 2, 3 -di oxygenase (IDO) inhibitors that interfere in this metabolic immune surveillance pathway. Our data show that a doxorubicin (DOX) encapsulating nanocarrier provides a more potent ICD stimulus than the free drug, and can do so synergistically with a small molecule inhibitor (indoximod) of the IDO-1 pathway. The nanocarrier is capable of facilitating this task by improving the PK of DOX and indoximod (IND) at the tumor site. This provides us with a first generation nanocarrier providing and ICD stimulus and an IDO inhibitor as a promising synergistic immunotherapy platform for BC, including triple negative BC (TNBC) (most responsive to immune checkpoint inhibitors) as well as ER-positive tumors (numerically the largest BC subtype responsible for mortality). RESULTS
Doxorubicin is an ICD-inducing chemoagent in breast cancer
[0767] In addition to improved intratumor drug content, we envisage the use of nanocarriers to deliver chemotherapy with a view to also implement BC immunotherapy. One possible approach is to use chemotherapy to induce immunogenic cell death or ICD. Consensus guidelines have been developed is to identify drug and chemo agents that can trigger ICD in vitro and in vivo (Kepp et al. (2014) Oncolmmunol. 3 : e955691). This allowed us to identify doxorubicin (DOX) as a potent ICD inducing chemotherapeutic agent for BC immunotherapy, using 4T1 cells in a syngeneic BALB/c vaccination model. Multi-parameter cellular screening demonstrated that DOX can induce the complete set of cell biological responses that lead to ICD, namely stress-induced cell death (calreticulin or CRT expression), ATP release (autophagy), and nuclear disintegration with release of HMGB1 (Fig. 10). In addition to DOX, we also identified paclitaxel (PTX) as an ICD inducer in 4T1 cells. In contrast, cisplatin (Cis) and 5-FU failed to induce the same effect. In vivo confirmation of the ICD-inducing effects of DOX and PTX was confirmed by a vaccination approach in immunocompetent BALB/c mice (Fig. 10). This involves subcutaneous (SC) injection of dying 4T1 cells, exposed to DOX (10 μΜ) or PTX (10 μΜ) for 24 h, into one flank of the animals, on two occasions, one week apart. The animals were re-challenged by injection of live 4T1 cells in the contralateral flank 7 days later (Fig. 10, panel B). The results
demonstrated that while vaccination with DOX- or PTX-treated cells could significantly suppress tumor growth at the challenge site, the negative control, Cis (50 μΜ), had no effect (Fig. 10, panels B, C). Additional in vitro ICD profiling (HMGB 1 and ATP release as well as CRT cell surface visualization) has been shown in our supporting information {see, e.g., Fig. 19). In the vaccination experiment in Fig. 10, bright field pictures of the excised tumors from the mice from each groupwere provided, in addition to the the averaged tumor weight data in Fig. 20. Moreover, bioluminescence visualization of 4T1 tumor development in the vaccination experiment was performed using IVIS imaging at different time points (Fig. 20). Mice body weight monitoring was provided (Fig. 20).
[0768] At the conclusion stage of this experiment, flow cytometry and IHC analyses were performed for immunophenotyping. IHC revealed increased tumor staining for CD8+ T cells in parallel with a decreased regulatory (Foxp3+) T cell component in animals vaccinated with DOX or PTX-treated cells (Fig. 21). Cis treatment had no effect. Quantitative assessment of the same biomarkers using flow cytometry demonstrated ~6- and ~5.5-fold increase in the CD8+/Tregs cell ratios in the DOX and PTX vaccinated groups, respectively, compared to saline (Fig. 21). CD8+/Tregs value for Cis group is -1.2. Moreover, IHC staining demonstrated that CD8+ T cells tumor infiltration was markedly increased in DOX and PTX-treated mice (Fig. 21). The number of Foxp3+ Tregs was significantly decreased compared to saline control. We also found that IFN-γ, Granzyme B, Perforin, and Cleaved Caspase-3 (CC-3), as well as CD103, CD80/CD86, and IL-12p70 levels were all drastically enhanced in DOX-treated group, which are indicative of the improved adaptive and innate immunity. PTX as a positive control had a similar effect on these immune markers as DOX.
Synthesis of a DOXIL® look-like DOX-laden IND-Liposome (DOX/IND- Liposome).
[0769] DOXIL® is a PEGylated liposome for the delivery of DOX and has been in the marketplace for two decades. Encapsulated DOX delivery holds significant advantages over free DOX in patients with Kaposi' s sarcoma, ovarian carcinoma and BC (Barenholz et al. (2012) J. Control. Re I. 160: 1 17-134). This advantage is in part derived from the improved PK of DOX at the tumor site as well as a reduction in cardiovascular and systemic DOX toxicity (Id.). DOX is loaded into DOXIL® by using a trapping agent, which generates a proton gradient that allows the import of weak-basic DOX through the liposomal lipid bilayer. One potential downside of DOXIL® is the preferential concentration of DOX in the skin, which can result in the hand-foot syndrome (redness and inflammation) (Id.). Clinical guidelines to avoid this side effect by adapting the DOXIL® dosing schedule exist. Against this background of this FDA-approved technology, we asked whether it was possible to develop a liposome for dual DOX and indoximod (IND) delivery. In addition to the improving the PK of DOX, we hypothesized that we would also be able to improve the circulatory half-life (Tv2) and tumor levels of indoximod (IND). This challenge was met by using bio-conjugation chemistry to synthesize a phospholipid-conjugated IND prodrug (IND- PL), as illustrated in Fig. 1 1, panel A. IND (D-l -methyl tryptophan or D-1MT) is covalently linked to l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PL) by an ester bond. An amazing feature of the prodrug is the self-assembly of IND-PL into 80 nm nanovesicles in an aqueous buffer. [0770] Briefly, to construct the DOX/IND-liposome, a classic DOX remote loading strategy was employed using ammonium sulfate as gradient (Fig. 1 1, panel B). The synthetic scheme was shown in Fig. 1 1, panel A. Following the evaporation of organic solvent in which contains IND-PL, Cholesterol, and DSPE-PEG2K, a uniform lipid film was formed along the bottom of the round flask. A protonating agent (NH4)2S04 was added into the flask afterwards, followed by probe sonication and PD-10 desalting column purification to render the pure (NH4)2S04-loaded IND-Liposome. Then DOXHCl solution was incubated with ( H4)2S04-loaded IND-Liposome at 65 °C for the active loading of DOX into the hydrophilic pocket of IND-Liposome.
[0771] DOX, a weak basic molecule, could easily be loaded into the liposome by using ammonium sulfate as a protonating agent in the self-assembly solution (Fig. 11). The proton gradient allows amphiphilic DOX to be imported across the liposomal membrane. This provided a dual drug delivering liposome that visually resembles DOXIL® (Figs. 11, panels C, D). To determine the optimal DOX/IND-Liposome, a mixture of lipids containing varied molar ratios of IND-PL, Cholesterol, and DSPE-PEG2K was tested when encapsulating a fixed amount of DOX. We found that IND-PL/Cholesterol/DSPE-PEG2K at molar ratio of 75:20:5 conferred the most stable and relatively smaller-sized nanoparticle (see Tables 6 and 7, below). Table 6. Development and optimization of DOX/IND Liposome nanoformulation.
Development and optimization of DOX/IND-Liposome. Optimal lipid compositions were systemically investigated by varying the molar ratios of IND-PL. Cholesterol, and DSPE- PEG2K, in which the ratio at 75% IND-PL, 20% Cholesterol, and 5% DSPE-PEG2K rendered the relatively high DOX loading with most formulation stability.
Figure imgf000157_0001
Table 7. Dynamic light scattering (DLS) characterization of DOX/IND-Liposome (total lipids: 50 mg, DOX: 5.7 mg, molar ration: IND/DOS 4.3/1). DLS characterization (size, PDI and zeta potential) of DOX/IND-Liposome in different media for 30 days.
Figure imgf000157_0002
Liposome (nm) (mV)
DI Water 102.4±3.56 0.10±0.01 -17.35±2.11 After 30 days 106.8±2.76 0.11±0.02 -18.63±2.98
PBS 103.2±1.72 0.09±0.01 -12.42±1.65
After 30 days 109.3±3.84 0.12±0.02 -11.87±0.75
10% FBS in DMEM 105.9±1.52 0.12±0.02 -19.21±1.52 After 30 days 116.2±2.03 0.14±0.01 -19.75±1.83
[0772] This ratio was, thereby, utilized for the subsequent DOX/IND-Liposome preparation for in vitro and in vivo assays. In this formulation, the drug loading for IND and DOX were 19.8% and 11.4%, respectively, with IND/DOX molar ratio at 4.3/1, determined by UPLC-MS/MS analysis. The DLS size is around 100 nm with narrow polydispersity and slightly negative charge. This optimal DOX/IND-Liposome is able to maintain its stability without significant size change for up to one month in DI water, PBS, and 10% FBS- containing DMEM. In addition, DOX/IND-Liposome mirrors that of DOXIL®, in terms of drug loading, size, morphology, as well as the charge (Fig. 11, panel C). Both of DOX formulations had accepted endotoxin level (< 0.1 EU/mL), which favors its potential clinical translation. Hydrodynamic size (DLS) and morphology (cryoEM) of DOX/IND-liposome and DOXIL® were provided in Fig. 11, panel D.
DOX IND-Liposome displays superior pharmacokinetics (PK) and tumor accumulation to free DOX: the pharmacokinetics is comparable to DOXIL®.
[0773] The DOX/IND liposome was used to assess its effects on PK and
biodistribution in an orthotopic BC model, established by injecting luciferase-transfected 4T1 cells into the mammary fat pad. The animals developed primary tumor nodules in the breast, with metastases to the lung -30 days post-implantation (Pulaski et al. (2000) Curr. Protoc. Immunol. 39: 20.2.1-20.2.16; Lu et al. (2014) Mo/. Pharmaceut. 11 : 4164-4178). In brief, orthotopic BC model was established by injecting 4T1-Luc cells (in 30 uL DMEM/matrigel, 1/1, v/v) into the right second mammary fat pat of Balb/c mice (Fig. 12, panel A). Two weeks after inoculation, a solid tumor mass was discerned via IVIS imaging. Dissection of the major organs revealed the detectable lung metastasis in addition to the primary tumor. Four weeks later, primary tumor grew up rapidly with severe lung metastasis.
[0774] To postulate the therapeutically relevant and safe DOX dose that is to use in vivo, first, we carried out a maximum tolerated dose (MTD) investigation following a protocol established by National Cancer Institute by IV injecting free DOX, DOX/IND- Liposome, and DOXIL® to Balb/c mice. The MTD was found to be 8, 15, and 15 mg/kg for DOX, DOXIL®, and DOX/IND-Liposome, respectively. Clinically, DOXIL® is IV administered at a dose of -50 mg/cm2 once a month. This allows us to use formula to convert the human dose into mouse dose. A human DOX dose of 50 mg/cm2/month equals to DOX mouse dose of -16.4 mg/kg per month. Based on MTD and clinical DOXIL® dose, we decide to use 5 mg/kg for 3 times IV injection in about one month period for the following PK and efficacy studies (Fig. 12, panel B).
[0775] Since DOX is a fluorescent molecule, the first experiment is to use IVIS image to monitor DOX tumor fluorescent signal after IV injection of the liposomes, delivering 5 mg/kg DOX and 15 mg/kg IND (n=3) (Fig. 12, panel C). Free DOX and DOXIL® were served as controls. 24 h post injections, major organs were collected and DOX fluorescence was visualized by IVIS imaging using excitation filter: 500 nm and Emission filter: DsRed, and quantified by Living Image® software (PerkinElmer, version 4.5). Tissue fluorescence at 0 h was obtained before drug injections as a reference. Compared to the weaker DOX tumor fluorescence observed in free DOX group, DOX/IND-Liposome markedly enhanced DOX tumor uptake by -10-fold, which is in agreement with that of DOXIL® (Fig. 12, panel C). [0776] Moreover, a comprehensive PK study was also performed. Orthotopic 4T1 tumor mice (n=6) were IV injected by free DOX, DOXIL®, and DOX/IND-Liposome at equivalent 5 mg/kg DOX. At predetermined time points (0.08333h, 0.25h, 0.5h, lh, 2.5h, 8h, 24h, and 48h) post IV injection, blood was withdrawn, and the plasma was acquired for quantitation of IND, IND-PL, as well as DOX via UPLC-MS/MS. Data calculation with WinNonlin software demonstrated a significant increase in the plasma ti/2 and intratumor levels of DOX compared to the free drugs. Free DOX was eliminated from the blood circulation rapidly by the mononuclear phagocytic system (MPS) due to its unprotected nature. Within 5 min post injection, more than 95% of free DOX disappeared; while the circulatory ti/2 of free DOX was extended from <0.083 h to -3 h by DOXIL® and the
DOX/IND liposome. Calculated as a % of the total injected dose, up to ~10 wt% (dose/g tissue) encapsulated DOX could be seen to distribute to the 4T1 tumor site by 48 h, as compared to -0.6% using free DOX (Fig. 12, panel D). We also demonstrated that the circulatory ti/2 of free IND was prolonged to a similar degree following encapsulation into the liposome and IV administration. UPLC/MS measurement of the IND content in the tumor also demonstrated an increase from 0.6 to 9.6 wt% for free versus liposomal delivery. The accordant PK and BD of DOX and IND in DOX/IND-Liposome upholds the notion that these two drugs were associated together during circulation. DOX IND-Liposome outperforms DOXIL® in controlling the orthotopic 4T1 breast cancer growth in mice.
[0777] We performed a comprehensive efficacy study to show the outcome of the anti -tumor immune response in the orthotopic 4T1 model. The co-delivery liposome as well as comparative controls are listed in Table 8.
Table 8. Treatement groups.
Figure imgf000160_0001
[0778] For ease of data presentation, the Is set of comparison includes the following treatments: #1 Saline, #2 DOX, #3 DOXIL®, #4 IND-Liposome, #5 DOX + IND-Liposome and #1 DOX/IND-Liposome. To compare the combination anti-BC outcome to anti-PD-1, the 2nd set of comparison includes #1 Saline, #6 DOX + anti PD-1, #7 DOX/IND-Liposome and #8 DOX/IND-Liposome + anti PD-1. To confirm the anti-tumor immunity of DOX/IND- liposome, we also included the treatment using "#10 DOX/IND-Liposome + anti CD8". Animals received IV injection of the same dose of encapsulated DOX and IND on three occasions, ~4 days apart (Fig. 14). The injected DOX dose is l/3rd of the pre-determined MTD dose (15.6 mg/kg) and comparable to the extrapolated dose of DOXIL® in animal studies. Our results demonstrated that the DOX/IND liposome induced higher rates of tumor shrinkage compared to the controls, including DOXIL® (Fig. 14, panels B,C). Orthotopic 4T1 breast tumor has been reported to predominantly metastasize to lung. To elucidate if DOX/IND-Liposome can simultaneously minimize the lung metastasis, mice were sacrificed and lung tissues were imaged by IVIS imaging system. We performed quantitative ex vivo IVIS imaging of the metastatic lung burden, which showed a highly significant reduction in secondary spread to this organ in response to DOX/IND co-delivery compared to DOXIL® (Fig. 14, panel D). Kaplan-Meier plots confirmed that the dual delivery liposome resulted in a significant survival benefit, including in comparison to DOXIL® (Fig. 14, panel E). Combination with immune checkpoint blockade leads to synergistically enhanced antitumor efficacy.
[0779] Immune checkpoints such as PD-1/PD-L1, and CTLA-4/B7 play an inhibitory role in tumor immune surveillance. Recent clinical trials using antibody inhibitors (anti PD- 1, anti PD-L1, and anti CTLA-4) targeting these pathways have achieved very promising patient survival benefits in many types of cancer, including certain BC scenarios. We asked if our DOX/IND liposome can synergize with checkpoint inhibitor, i.e. anti-PDl . We demonstrated that PD-1 was highly expressed in 4T1 breast tumor tissues via
immunohistochemistry (IHC) (Fig. 15, panel A); this justifies the combinatorial application of anti PD-1 antibody with DOX/IND-Liposome. Concurrent therapy of anti PD-1 synergistically reinforced the DOX/IND-Liposome-induced tumor control (Fig 15, panels B- D). Surprisingly, the 4T1 lung metastasis has been completely prevented by this combination therapy. Eventually, the augmented antitumor efficacy and depleted tumor metastasis gave rise to the significantly further prolonged mice survival (Fig. 15, panel F). Improved DOX IND-Liposome antitumor effect involves the activation of adaptive and innate immunity, as well as stimulation of mTOR pathway.
[0780] It is extrapolated that the augmented tumor suppression by our dual delivery
DOX/IND-Liposome could be ascribed to its potential to make dual use of DOX-induced ICD and IND-PL-mediated IDO pathway inhibition. To validate the hypothesis, we also include a control treatment (#9) to deplete CD8+ T cells via intraperitoneal (IP) injecting anti CD8 antibody 3 days prior to the first DOX/IND-Liposome IV injection. IP administration of anti-CD8 in this group was continued every 2-3 days until the end of the animal study. This allows us to demonstrate a significant interference on co-delivery liposome anti-cancer effect. Upon depletion of CD8 T cells systemically, DOX/IND-Liposome antitumor activity was drastically hampered with recovered lung tissue metastasis, which invariably resulted in shortened breast cancer mice survival rate (Fig. 16).
[0781] For all the treatments in Table 8, we performed comprehensive
immunophenotyping of the primary tumor TILs, using IHC staining and multi-parameter flow cytometry (Fig. 17). The tumors were isolated at the end of the efficacy study and were subject to the flow cytometry and IHC for a variety of different immune biomarker analyses. Flow data showed that CD8/Treg ratios were markedly increased in DOX/IND-Liposome- treated mice compared to other controls and DOXIL®, while anti PD-1 checkpoint blockade further uplifted this ratio. In a sharp contrast, depletion of systemic CD8 resulted in an almost completely reduced CD8/Treg ratio, in a manner that is similar to the saline control group (Fig. 17). IHC staining for CD8 and Foxp3 was well in line with the flow results, where CD8+ T cells tumor infiltration was significantly improved with decreased number of Foxp3+ Treg in DOX/IND-Liposome group. When combing with anti-PD-1, the CD8/Treg ratio was further increased (Fig. 17). Consistent results were unearthed for IFN-γ, granzyme B, peforin, and CC-3 by either multi-color flow cytometry or IHC (Figs. 17 and 22). These data substantiate the claim that adaptive immunity played a pivotal role in contributing to the overall tumor reduction of DOX/IND-Liposome. [0782] Apart from the adaptive immune response, innate immunogenicity was also investigated. Both the CRT expression and its corresponding receptor CD91 on DCs were remarkably uplifted in our dual delivery particle-doped group, which were further enhanced with addition of anti PD-1, and attenuated drastically by co-injection of anti-CD8 antibody (Fig. 17 and 22). CD80 and CD86, found on DCs, work in tandem to provide a costimulatory signal necessary for T cell activation and survival. Herein, we found that the expression for CD80/CD86 was noticeably increased in DOX/IND-Liposome. Combination with anti PD-1 therapy furthered this improvement. This trend was also identified in the expression of CD 103, an integrin on DCs that is known to facilitate the CD8+ T cell development and stimulation (Fig. 17). Also, IL12p70, a naturally produced interleukin by DCs and
macrophages, and involved in the differentiation of naive T cells into Thl cells that stimulates the growth and function of T cells, was substantially boosted in our dual
DOX/IND-Liposome. Additional low magnification IHC data is provided on Fig. 22. Taken together, innate immune response was actively engaged in the antitumor immunity aroused by DOX/IND-Liposome. Safety assessment of DOX IND liposome in mice
[0783] From a drug safety perspective, the dual delivery liposome, similar to
DOXIL®, showed normalization of the elevated levels of troponin I and creatine kinase from the heart, liver alanine aminotransferase and aspartate aminotransferase from the liver and creatinine retention by the kidney, compared to free DOX. Moreover, the dual-delivery DOX/IND-Liposome was well tolerated as manifested by no weight loss, increased cardiac troponin I, creatine kinase, ALT, AST, as well as creatinine (Fig. 18). In a sharp contrast, goups involving free DOX resulted in severe toxicity as shown by significantly elevated biomarkers for heart, liver and kidney toxicity. Methods
Cell and mice.
[0784] 4T1 cell line, a highly aggressive phenotype similar to that observed in human triple-negative breast cancer, grows and metastasizes in Balb/c mice closely mimic human breast cancer. This tumor is an animal model for stage IV human breast cancer. 4T1 cell line was obtained from ATCC and used for the cellular studies and establishing subcutaneous and orthotopic tumors in mice. 4T1 cells were cultured in complete DMEM medium, containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine in 37 °C incubator with 5% C02. To realize the visualization of 4T1 tumor growth by IVIS bioluminescence imaging, 4T1 cells were stably transfected with a luciferase expressing lentiviral vector in the vector core facility at UCLA. Female Balb/c mice (Charles Rivers, 6-8 weeks old) were used to establish subcutaneous or orthotopic 4T1 tumors. Mice were housed under pathogen-free conditions and all animal experiments were approved by the UCLA Animal Research Committee. CRT surface localization, and HMGB-1 and ATP release.
[0785] l x lO5 4T1 cells were plated in 24-well plates overnight in complete DMEM medium. The cell culture medium was aspirated and replenished with PBS (Ctr), Cis, DOX, PTX and OX containing media at various concentrations for 24 h. 100 uL supernatants were obtained for HMGB-1 or ATP measurement using an HMGB-1 ELISA kit (IBL International GmbH) or ATPlite 1-step Luminescence Assay Kit (PerkinElmer) following the
manufacturer's instructions. To determine the CRT surface expression, 4T1 cells were first trypsinized, and washed in cold PBS for three times. Then cells were stained with a primary rabbit anti-CRT antibody (Ab2907, Abeam), followed by an Alexa Fluor® 680-conjugated goat-anti rabbit IgG antibody for 30 min at 4 °C. The stained cells were suspended in 500 μΕ staining buffer (BD Biosciences), in which 50 μg/mL propodium iodide was included prior to washing and measurement in a LSRII flow cytometer (BD Biosciences). Flow cytometry data were plotted as fold-change in mean fluorescence intensity (MFI) in comparison to the PBS control. The experiment was repeated twice. To visualize the CRT translocation to 4T1 cell surface, 1 >< 1044T1 cells were seeded in Lab-Tek® 8-well Chamber slides (Thermal Scientific) for overnight, and then treated with Cis (100 μΜ), DOX (1 μΜ), PTX (1 μΜ) and OX (50 μΜ) for 24 h, respectively. Cells were fixed in 4% PFA for 15 min and then washed with cold PBS three times before being stained with an Alexa Fluor® 647-conjugated anti- CRT antibody (abl96159, 1/500, Abeam) for 30 min. To illuminate the cell surface membrane, cells were subsequently doped with 5 μ§/ιηΙ. Alexa Fluor®488-conjugated wheat germ agglutinin (WGA, W32466, 1/200, Thermo Fisher). Finally, cells were stained with Hoechst 33342 nuclear dye (H3570, 1/2000, Invitrogen) and subject to the observation under a Leica SP8-SMD confocal microscope (63 χ objective lens).
Intracellular uptake of IND-Liposomes in 4T1 cells.
[0786] l x lO5 4T1 cells were seeded in 24-well plates overnight in complete DMEM medium. The next day the cells were treated with free IND or IND-NV at equivalent 100 μg/mL IND dose for 4, 24, and 72 h, respectively. Cells were then trypsinized, collected and dissolved in methanol overnight for drug concentration measurement using UPLC-MS/MS following our established protocol.
Mechanistic targeting of niTOR and GCN2 by the IND-Liposome.
[0787] 1 x 106 4T1 cells were plated into each well in 6-well plate in tryptophan- deficient DMEM (Gibco). After overnight culture, cells were challenged with IND or IND- NV for 3 h at 10 uM and 50 uM. The supernatants from the same experiments were gleaned for the IL-6 determination by an ELISA kit (BD Biosciences). In addition, western blotting was performed to assess the P-S6, total S6K and GAPDH protein levels as previously.
Briefly, total protein was extracted from 4T1 cells which were then lysed in RTPA lysis buffer supplied with proteinase and phosphatase inhibitor cocktail. After centrifugation of cells lysate at 12,000 RPM for 10 min, equal amount of total protein samples in supernatants was loaded into 10-20% tris-glycine gel (Novex gel, Invitrogen) and transferred to PDVF member subsequently. Membrane was first blocked with 5% BSA/TBST and incubated with primary and HRP conjugated secondary antibodies. The blots were detected using with ECL substrate (Thermo Scientific), exposed to film and developed. To investigate the effect of IND-NV on IDO pathways in vivo, tumors were cut into small pieces and homogenized in RTPA lysis buffer supplied with proteinase and phosphatase inhibitor cocktail, and then centrifuged at 12,000 RPM for 10 min. The proteins in supernatants were subject to the procedures as described above. The intensity of bands for each protein on the film was quantified by Image J software (Lu et al. (2017) Nat. Comm. 8: 1811). Vaccination using dying 4T1 cells in Balb/c mice.
[0788] First, 4T1 cells were treated with PBS, 100 μΜ Cis, 50 μΜ OX and 1 μΜ
DOX for 24 h. The cell suspensions were then collected and subject to the CRT surface expression verification by flow cytometry. Afterwards, 1 x 106 dying 4T1 cells in 0.1 mL of DMEM were administered into the right flank of female Balb/c mice (n=6), at one week interval. Two weeks post the 1st vaccination of 4T1 cells, the same mice were SC injected by healthy 1 x 106 4T1 cells in 0.1 mL DMEM/matrigel, 1/1, v/v at the left flank. Tumor size was closely monitored every 3-4 days by a digital caliper, and the size was calculated based on the formula: π/6 χ length χ width2. Besides, IVIS imaging was carried out on day 10, 16, and 19 to render the visualization of the tumor burden. "Spaghetti plots" detailing each individual mouse tumor growth were plotted. Mice were sacrificed on day 19 and the tumors were collected for flow cytometry and IHC analysis, both of which are detailed below.
Statistical difference among groups was compared by two-way analysis of variance (SPSS software). On day 19, tumors were harvested for the analysis of flow cytometry and IHC, both of which were detailed in the below sections.
Synthesis of the DOX/IND-Liposome.
[0789] First, the IND-PL conjugate was synthesized following the published method
(Lu et al. (2017) Nat. Comm. 8: 1811). Three steps were involved: 1) protection of the amine group on IND by coupling it with the Di-tert-butyl dicarbonate (Boc anhydride); 2)
Conjugating the OH- on PL to COOH- on IND via esterification; 3) Removal of the Boc group to render the active IND-PL prodrug. The final IND-PL was verified by lH-NMR, 13C-NMR and ESI-MS. DOX-laden IND-Liposome was prepared through remote loading using ammunium sulfate as a gradient, in which the lipid bilayer is composed of the IND-PL. 50 mg lipid mixture containing 75% IND-PL. 20% Cholesterol, and 5% DSPE-PEG2K (molar ratio) was dissolved in chlorofrom in a 50 mL round bottom glass flask. This ratio was derived by experimenting with different lipid mixtures (Fig. 22). The organic solvent was removed via a rotary evaporator, leading to the formation of a thin lipid film, which was then placed in vacuum overnight for further dry. The lipid film was then hydrated with 2 mL of (NH4)2S04 (123 mM) and probe sonicated for 30 min using a 20/15 second on/off cycle at a power output of 32.5W. The free (NH4)2S04 was removed via passing through the
Sephadex G-25 PD-10 Desalting Column (GE Healthcare) using PBS as eluting solvent. To achieve uniformly-sized IND-Liposome, the nanoparticle-containing solution was extruded 15 times through a Mini -Extruder (Avanti Polar Lipids), with a 100 nm pores polycarbonate membrane (Avanti Polar Lipids) at 80 °C. Afterwards, the 6.47 mg DOX (10 mg/mL) was added into the above purified ( H4)2S04/IND-Liposome solution, and incubated for 30 min at 65 °C. The resultant mixture was subsequently subject to the Sephadex G-25 PD-10 Desalting Column purification to remove the free DOX to deliver the DOX/IND-Liposome. The purified DOX/IND-Liposome was comprehensively characterized for size, zeta potential, loading capacity, morphology, and endotoxin level using DLS, UPLC-MS/MS, cryoEM and the Chromogenic LAL Assay, respectively. The optimal particle batch was composed of nanoparticles with size -100 nm, slightly negative charge and at least one month formulation stability. Nanoparticles were kept at 4°C and away from the light prior to in vitro and in vivo applications.
Establishment of the orthotopic 4T1 tumor model.
[0790] 1 106 4T1 cells in 30 μL· DMEM/matrigel, 1/1, v/v, were injected to the right
2nd mammary fat pad of female Balb/c mice, 6-8 weeks old. IVIS imaging confirmed the successful orthotopic 4T1 tumor development within two weeks. After 3-4 weeks, predominant tumor metastasis was found at lung tissue, which is consistent to the literature.
Pharmacokinetics (?K) and biodistribution (BD) of systemically delivered DOX formulations.
[0791] 4T1 orthotopic tumor bearing mice were established as described above. For
BD of DOX formulations, free DOX, DOXIL®, and DOX/IND-Liposome were IV injected at equivalent 5mg/kg DOX to mice (n=3). 24 h post administration, mice were sacrificed and ex vivo DOX fluorescence images were procured for the extracted tumor, heart, liver, spleen, lung, and kidneys by IVIS imaging (Excitation filter: 500 nm; Emission filter: DsRed).
Tissue reference fluorescence was obtained before drug injections (0 h). DOX fluorescence was quantified by Living Image® software (PerkinElmer, version 4.5). For PK study, orthotopic 4T1 tumor mice (n=6) were received a single IV injection of free DOX, DOXIL®, and DOX/IND-Liposome at equivalent 5 mg/kg OX. At 0.08333h, 0.25h, 0.5h, lh, 2.5h, 8h, 24h, and 48h post IV injection, 50 μΕ of blood was drawn, and the plasma was acquired and then digested in methanol overnight prior to UPLC-MS/MS analysis for IND, IND-PL, as well as DOX. In a separate study, 24 h post IV administration of DOX formulations, the tumor, heart, liver, spleen, lung, and kidney were excised and digested in methanol for 48 h before the determination of IND, IND-PL, as well as DOX using UPLC-MS/MS. The UPLC-MS/MS condition for measuring IND and IND-PL was well documented in our published paper (Lu et al. (2017) Nat. Comm. 8: 1811). For DOX quantification, UPLC- MS/MS setting was as follows: a C18 Column (13θΑ, 1.7 μπι, 2.1 mm x 50 mm), which was connected to Waters LCT Premier with ACQUITY UPLC and Auto sampler; gradient elution utilized: (i) 0 - 4 min, 95% water + 5% Acetonitrile; (ii) 4 - 6.5 min, 5% water + 95%
Acetonitrile; and (iii) 6.5 -10 min, 95% water + 5% Acetonitrile. The flow rate was 0.4 L/min. The t\a of DOX formulations was calculated according to a non-compartmental model or a two compartment model via WinNolin software.
Therapeutic efficacy investigation of the DOX IND-Liposome.
[0792] 1 106 4T1 cells in 30 μL· DMEM/matrigel, 1/1, v/v, were injected to the right 2nd mammary fat pad of female Balb/c mice. 8 days later, when tumor size reached 100-150 mm3, orthotopic 4T1 tumor-bearing mice (n=9) were randomly distributed to 9 groups. Mice were IV injected with DOX/IND-Liposome at a dose of 5 mg/kg DOX and 19.4 mg/kg IND on day 8, 11, and 14 for a total of three injections. Controls contain Saline and equivalent doses of free DOX, DOXIL®, IND-Liposome, and DOX + IND-Liposome, which were IV administered at the same frequency. Immune checkpoints such as PD-1/PD-L1 axis have been found to negatively regulate T activation with enhancement on Treg, leading to the tumor immunosuppressive microenviroment. To unleash the intrinsic tumor immunity and maximize the overall antitumor efficacy, anti PD-1 antibody was used in combination with DOX/IND-Liposome. We also included DOX + anti PD-1 group as a corresponding control. In either group, anti-mouse PD-1 (clone: RMP1-14, BioXcell) was IP injected at 100 μg/mouse on day 8, 11, and 14. To elucidate whether the tumor growth inhibition of DOX/IND-Liposome was attributed to the wakened systemic CD8 T cell immunity, systemic knockdown of CD8 was realized by IP administering anti-mouse CD8A mAb (clone 53-6.72, 200 μg/mouse, BioXcell) along with DOX/IND-Liposome. The injection of anti CD8 mAb commenced 3 days prior to the first IV injection of DOX/IND-Liposome and was carried on every 2-3 days until the end of the animal study. The CD8 T cell depletion was confirmed by immunohistochemistry (IHC). Tumor development was carefully monitored by digital caliper on day 8, 11, 14, 17, and 22. In addition, to visualize the tumor burden
bioluminescence, mice were imaged by IVIS imaging system after being injected IP with 75 mg/kg D-Luciferin. At the end of the study, the tumors and lungs were garnered for the visualization of the primary tumor mass, and the quantification of ex vivo bioluminescence image intensity of metastatic tumor spread in lung. Operator-defined area of interest (ROI) intensity in lung tissues was quantified by Living Image® software (PerkinElmer, version 4.5). In addition, tumor tissues were evenly divided for flow cytometry, IHC analysis, as well as the western blotting for P-S6K protein and RT-PCR for IL-6 mRNA. Further, the blood was drawn from mice for the evaluation of heart toxicity (cardiac troponin I and creatine kinase), liver damage (ALT and AST), and nephropathy (creatinine) associated with the administration of different treatments. To glean the mouse survival outcome, the animal efficacy study was repeated once following the exact same procedures and treatments (n=9). Mouse survival rate was displayed as Kaplan-Meier plots.
Immunohistochemistry (IHC).
[0793] During the innate and adaptive immune responses, the phenotypes of various immune cell populations may have been modulated. To visualize this change, IHC at the tumor sites was conducted as published previously (Lu et al. (2017) Nat. Comm. 8: 1811). Briefly, first, tumor pieces were fixed in 10% formalin, then embedded in paraffin and cut into 4 μπι thickness sections that were subsequently mounted on glass slides by UCLA Jonsson Comprehensive Cancer Center Translational Pathology Core Laboratory for IHC staining of CD8, Foxp-3, CRT, CD91, IL12p70, CC-3, LC-3, IDO and PD-1. Then, the slides were deparaffinized, incubated in 3% methanol-hydrogen peroxide, prior to immersing in 1 mM sodium citrate (pH=6) or 10 mM EDTA (pH=8) at 95°C in the Decloaking NxGen Chamber (Biocare Medical, DC2012). The slides recovered at room temperature, then rinsed by Phosphate Buffered Saline containing 0.05% Tween-20 (PBST). Afterwards, the slides were incubated with different primary antibodies for 1 h followed by incubation with corresponding HRP-conjugated secondary antibodies at RT for 30 minutes. For visualization of different immune cells, the slides were incubated with Vulcan Fast Red Chromogen Kit 2 (CD91/LRP1 only) (Biocare Medical, FR805) or DAB (3,3'-Diaminobenzidine, the rest of remaining). Finally, after tap water rinse, the slides were counterstained by Harris'
Hematoxylin, dehydrated in ethanol, and mounted with media prior to scanning in Aperio AT Turbo Digital Pathology Scanner (Leica Biosystems). The IHC data was rigorously analyzed by a veteran pathologist. Primary antibodies: Anti-CD8 (#14-0808, 1/100), and anti-Foxp3 (#13-5773, 1/200) were from eBioscience; anti-CRT (ab2907, 1/50), anti-LRPl(CD91) (ab92544, 1/50), anti-perforin (abl6074, 1/100) and anti-PD-1 (ab214421, 1/100) were from Abeam; Anti-cleaved caspase 3 antibody (#9664, 1/200) was from Cell Signaling; anti-IFN-γ (NBPl-19761, 1/200) and anti-IL12p70 (NBPl-85564, 1/100) was from Novus Biologicals; anti-LC-3 (0231-100/LC3-5F10, 1/100) was from Nanotools; anti-IDO (#122402, 1/100) was from Biolegend. Secondary antibodies: except CD91 that was incubated with MACH2 Rabbit AP -Polymer (Biocare Medical, RALP525), the remaining biomarkers were all incubated with Dako EnVision+ System HRP-labelled polymer Anti-Rabbit (Dako, K4003).
Flow cytometry.
[0794] Multi-parameter staining for cell suspensions was performed as published protocol. Briefly, tumors collected from vaccination and orthotopic efficacy studies were first cut into small pieces with knife (Personna) followed by digestion in collagenase type I (0.5 mg/mL, Worthington Biol Corporation) in Benchtop Incubating Shaker (MaxQ Digital 4450, Thermo Scientific) for 1 h at 37 °C. The resultant tissues were meshed though a 70 DM cell strainer twice with caution. The cell pellets were suspended in 5 mL Ack lysing buffer (Gibco) in 4 °C for 5 min to lysate the red blood cells. After centrifugation at 1,500 RPM for 5 min, the single cell suspensions were washed with cold PBS twice and then resuspended in staining buffer (554656, BD Biosciences). To block nonspecific binding, cell suspensions were incubated with FcBlock (TruStain fcX™ anti-mouse CD16/32, clone 93, BioLegend) for 20 min after aliquoting. Multi-parameter staining was performed by utilizing different combinations of fluorophore-conjugated antibodies for 40 min at 4 °C. Dead cells were excluded by 7-aminoactinomycin D (7AAD, Sigma); doublet cells were excluded based on forward and side scatter nature. The following immune cell subpopulations were
investigated: (i) CD8+ T cells (CD45+CD3+CD8+CD25+), (ii) Tregs
(CD45+CD3+CD4+Foxp3+), (iii) IFN-y+ T cells (CD45+CD3+CD8+IFN-Y+), (iv) Granzyme B+ T cells (CD45+CD3+CD8+Granzyme B+), (v) CD91+ DC-like cells
(CD45+CDl lb+CDl lc+CD91+), (vi) CD80+/CD86+ DCs (CD45+CD1 lc+CD80+CD86+), and (vii) CD103+ DCs (CD45+CD1 lb+CDl lc+CD103+). Anti -mouse antibodies sources are as follows: CD45-V450 (#560501, 1/100), CD45-APC-Cy7 (#557659, 1/100), CD4-Alexa Fluor488 (#557667, 1/100), Foxp3-PE (#563101, 1/100), CD8a-PE (#561095, 1/100), CDl lb-PE (55331 1, 1/100), and CD 1 lc-V450 (560521, 1/100) were from BD Biosciences; CD103-Alexa Fluor 647 (#121410, 1/250) and IFN-y-APC (505810, 1/100) were from BioLegend. LRP1 (CD91)-Alexa fluor 647 (ab l95568, 1/250) was from Abeam. CD3-APC- eFluor780 (#47-0032-82, 1/100), CD25-APC (#17-0251-82, 1/100) and Granzyme B-eFluor 660 (50-8898-82, 1/100) were from eBiosciences. To stain intracellular Foxp3, IFN-γ, and Granzyme B, cells were fixed and permeabilized using the Staining Buffer Set (00-5523-00, eBioscience) followed by PBS washing prior to flow cytometry determination (LSRII, BD Biosciences). The flow data were potted as normalized ratio change as compared to control after analysis by FlowJo software (Tree Star). Statistical Analysis.
[0795] Differences among groups were analyzed via analysis of variance (ANOVA);
Kaplan Meier survival curves were compared using the Log-rank Mantel-Cox test (version 23, SPSS). Results were presented as mean ± SEM of at least three independent experiments. Statistical significance were set at *p <0.05; **p <0.01; #p <0.001.
Example 4
DOX IND co-delivering by lipid bilayer-coated MSNPs (silicasomes).
[0796] Figure 6 illustrates the synthesis of DOX-laden IND-PL coated MSNP. A
DOX/IND-MSNP dual-delivery carrier was designed by trapping DOX in the mesoporous interior of a -65 nm MSNP, using the lipid bilayer into which IND-PL was incorporated. In order to apply the lipid coating, we used the previously described biofilm method for rapid encapsulation, by sonication (Meng et al. (2015) ACS Nano, 9(4): 540-3557; Liu et al. (2016) ACS Nano, 10: 2702-2715). DOX was then remotely loaded using the protocol as previously described (Id). This involves 3 steps: 1) Bare MSNPs: Bare MSNPs can be made by a sol-gel synthesis process as we shown before (Meng et al. (2015) ACS Nano, 9(4): 540-3557); 2), Including the trapping agent in the IND-PL coated MSNPs; 3) Remote DOX loading. A total of 50 mg lipids containing IND-PL/cholesterol/DSPE-PEG2K (molar ratio, 75:20:5) was dissolved in 5 mL chloroform in a 50 mL round bottom glass flask. The solvent was evaporated under a rotatory vacuum to form a uniform thin lipid film, which was further dried under vacuum overnight. 40 mg MSNP (40 mg/mL in DI water) was centrifuged and re-suspended in 2 mL ammonium sulfate (123 mM), followed by probe sonication for 5 min. The ammonium sulfate soaked MSNPs were then added to the IND-PL containing lipid biofilm, followed by 30 min probe sonication to derive the ammonium sulfate-loaded IND- PL coated MSNP. To remove the free ammonium sulfate, the particle suspension was passed through a PD-10 size exclusion column. Ammonium sulfate-containing IND-PL coated MSNPs were eluted from column faster than free ammonium sulfate due to its large size. Remote Dox loading and purification: 6.5-32.4 mg of DOX'HCl (10 mg/mL) in DI water was incubated with ammonium sulfate laden IND-PL coated MSNP at 65 °C for 40 min. The pure DOX/IND-MSNP was collected by centrifuging at 15,000 rpm for 15 min, three times. The purified DOX/IND-MSNPs were fully characterized for size, charge, loading capacity, morphology and endotoxin level using DLS, UPLC-MS/MS and ICP-OES, cryoEM and the Chromogenic LAL Assay, respectively. The final product was stored at 4°C in the dark prior to biological test. The hydrodynamic size of the DOX/IND-MSNPs was -110 nm. The IND and DOX loading capacities are tunable, ranging from 0-28.2 wt% and 0-75 wt%, respectively. The lipid thickness was about 6-7 nm.
Example 5
Efficacious Pancreas Cancer Immunotherapy using Nanocarriers to Induce Synergistic
Immunogenic Cell Death and Immunomodulatory Responses
[0797] While chemotherapy delivery by nanocarriers has modestly improved the survival prospects of pancreatic ductal adenocarcinoma (PDAC), additional engagement of the immune response could be game changing. We developed a nano-enabled approach for accomplishing robust anti-PDAC immunity in syngeneic mice through the induction of immunogenic cell death (ICD) as well as interfering in the immunosuppressive indoleamine 2,3-dioxygenase (IDO) pathway (see, e.g., Figure 24). This was accomplished by
conjugating the IDO inhibitor, indoximod (IND), to a phospholipid that allows the prodrug to self-assemble into nanovesicles (IND-NV) or to be incorporated into a lipid bilayer that encapsulates mesoporous silica nanoparticles (MSNP). The porous MSNP interior allows contemporaneous delivery of the ICD-inducing chemotherapeutic agent, oxaliplatin (OX). IND-NV plus free OX or OX/IND-MSNP induced effective innate and adaptive anti-PDAC immunity when used in a vaccination approach, direct tumor injection or intravenous biodistribution to an orthotopic PDAC site. Significant tumor reduction or eradication was accomplished by recruited cytotoxic T lymphocytes, concomitant with downregulation of FoxP3+ T-cells.
[0798] In this Example, we report the design of nanocarriers to facilitate the induction of ICD and interference in the Kynurenin pathway, either through the development of nanovesicle that delivers an IND pro-drug or a lipid-coated MSNP, which co-delivers phospholipid-conjugated IND plus OX. We demonstrate the feasibility of achieving tumor regression or eradication of Kras-induced PDAC tumors, grown subcutaneously or orthotopically implanted by using the nanocarriers for a vaccination approach, local tumor injection or systemic administration. Our data show that the synergy between ICD and interference in the IDO pathway boosts innate and adaptive immunity in the syngeneic animal KPC model. This leads to effective killing of pancreatic cancer cells by CD8+ cytotoxic T cells at the tumor site, as well as interfering in metastatic spread. The cytotoxic response is accompanied by disappearance of Tregs at the tumor site, in addition to evidence of boosting innate immunity through increased CRT and toll-like receptor 4 (TLR4) expressions. The systemic immune response could also be adoptively transferred to nonimmune animals.
Results
Oxaliplatin-induced ICD provides a successful vaccination approach for PDAC
[0799] ICD is a modified form of apoptosis that can be used to initiate an effective immune response against endogenous tumor antigens (Kroemer et al. (2013) Ann. Rev.
Immunol, 31 : 51-72). ] Since this model was 1st proposed against the backdrop of a select number of cancer drugs (Id.), we focused on the use of OX, because it is FDA-approved for PDAC treatment. As a component of the FOLFIRINOX regimen (in combination with irinotecan, 5-FU and folinic acid). For comparison, we also included the anthracycline antibiotic, DOX, as a positive control and cisplatin (Cis) as a negative control for the screening of PDAC cell lines, using cell surface CRT expression (Obeid etal. (2017) Nat. Med, 13(1): 54-61; Casares et al. (2005) J. Exp. Med. 202(12): 1691-1701; Fucikova et al. (2011) Cane. Res. 71(14): 4821-4833; Tesniere etal. (2010) Oncogene, 29(4): 482-491;
Galluzzi et al. (2012) Oncogene, 31(15): 1869-1883; Martins et al.(20\ \) Oncogene, 30(10): 1147-1158). CRT is an endoplasmic reticulum (ER) stress protein that translocates to the surface membrane of cancer cells undergoing ICD (Obeid etal. (2017) Nat. Med, 13(1): 54-61; Fucikova et al. (2011) Cane. Res. 71(14): 4821-4833). Screening for CRT expression was performed in murine KPC cells, derived from a spontaneous tumor that developed in a transgenic KrasLSL"G12D/+/Trp53LSL"R172H/+/Pdx- 1 -Cre (KPC) mouse (Hingorani et al. (2005) Cancer cell, 7(5): 469-483).
[0800] The KPC model recapitulates many of disease features of human PDAC, including oncogene expression, development of a robust cancer stroma, extensive local invasion and distant metastases (Torres et al. (2013) PloS one, 8(11): e80580; Tseng et al.
(2010) Clin. Cane. Res. 16(14): 3684-3695). Confocal microscopy demonstrated the presence of red fluorescence (ALEXA FLUOR® 647 labeled antibody) staining for CRT on the surface of OX and DOX-treated KPC cells (Figure 25, panel a). In contrast, negligible or no surface staining was seen in cells treated with Cis or PBS (Figure 25, panel a). More quantitative data were obtained by flow cytometry, demonstrating dose- and time-dependence for the responses to OX and DOX (Figure 25, panel b). This amounted to a 1.6- and 2.5-fold increase in CRT expression levels in cells treated with 200 μΜ OX at 4 and 24 h,
respectively (Figure 25, panel b, Figure 31, panel a). A similar stress response was observed in the human PANC-1 pancreatic cancer cell line (Figure 31, panel b), as well as measuring HMGB-1 release by ELISA in both cell types (Figure 31, panel c). FDVIGB-1 is released from the nucleus of dying cancer cells and plays an important role in boosting APC activity during the ICD response. [0801] To develop an animal model for testing the feasibility of OX-induced ICD being able to induce effective anti-PDAC immunity, we used KPC cells to grow
subcutaneous (SC) tumors in syngeneic, immune competent B6/129 mice. To allow bioluminescence imaging of the tumor site, the cells were transfected with a luciferase vector, as previously described by us (Liu et al. (2016) ACS Nano, 10(2): 2702-2715). The 1st experiment assessed whether ex vivo exposure of KPC cells to OX can ICD that can be tested in a SC vaccination approach. The cells suspensions were generated by exposing the KPC cells to OX (50 μΜ), DOX (1 μΜ), or Cis (100 μΜ), followed by SC injection on 2 occasions, 7 days apart, prior to re-challenging the same animals by SC injection of live KPC cells on the contralateral flank (Figure 25, panel c). While vaccination with OX- or DOX- treated cells significantly suppressed tumor growth on the contralateral side, Cis treatment had no effect (Figure 25, panel d). The magnitude of the growth inhibition was confirmed by IVIS imaging, which showed decreased bioluminescence intensity in the contralateral site for OX and DOX but not Cis (Figure 32, panel a). Notably, 3 (out of 7) mice in the OX-treated group and 2 (out of 7) mice in DOX-treated group survived tumor-free and could be used in subsequent re-challenge experiments (see below). The rest of the animals were sacrificed on day 29 for immunohistochemistry (IHC) and flow cytometry analysis of the tumor tissues.
[0802] IHC revealed increased staining intensity for CD8+ T cells in the resected tumors, in parallel with a decreased regulatory (Foxp3+) T cell component in animals vaccinated with OX or DOX-treated cells (Figure 25, panel e). Cis treatment had no effect. Quantitative assessment of the same biomarkers by flow cytometry in single cell suspensions, demonstrated a 5.1- and 5-fold increase in the CD8+/Tregs cell ratios in the OX and DOX vaccinated groups, respectively, compared to Saline (Figure 25, panel e, right panel). In contrast, the change in the Cis group was only 1.3-fold. Significantly fewer CD4+ T-cells were seen at the tumor sites of OX and DOX-treated groups (Figure 32, panel c). Since elevation of the CD8+/Tregs ratio is compatible with an effective cytotoxic response, IHC staining was performed to look for evidence of cell death and IFN-γ production by the newly recruited CD8+ cells. This analysis demonstrated the presence of intense staining for cleaved caspase-3 (CC-3) (Figure 25, panel f, right panel) and IFN-γ (Figure 25, panel f, left panel) in tumors, harvested from animals vaccinated with OX or DOX-treated cells.
[0803] The 3 surviving animals in the OX-induced ICD group were used for intra- pancreatic injection of live KPC cells on day 74. No orthotopic tumors emerged during the subsequent observation period (up to date, 132), compared to control (non-vaccinated) animals, which succumbed due KPC tumor development metastases within -30 days. The surviving, prior vaccinated and orthotopic-challenged animals were sacrificed on day 132 to harvest immune splenocyte populations for adoptive transfer to non-immune animals. This was performed by intravenous (IV) injection of the splenocytes into the tail vein of 12 non- immunized B6/129 mice. The control was a group of 12 animals receiving IV injection of splenocytes harvested from non-immune animals or Splenocytes obtained from 12 animals treated with saline only. Each of the 3 groups was divided in half, with 6 animals receiving SC injection of live KPC cells and the rest being injected with B16 melanoma tumor cells. Monitoring of the animals for tumor growth over 26 days, demonstrated a significant reduction in KPC growth in animals injected with immune splenocytes, compared to animals receiving non-immune splenocytes or saline only (Figure 25, panel g). 2 of the 6 mice receiving immune splenocytes survived tumor-free. In contrast, there was no impact of immune or non-immune splenocytes on B16 tumor growth (Figure 33). All considered, these results indicate that OX treatment is effective for generating ICD that results in effective anti- PDAC immunity. Moreover, this response generates an antigen-specific, cognate immune response that can be adoptively transferred to recipient animals.
Synthesis of a self-assembling indoximod (IND) pro-drug for immunomodulatory therapy
[0804] IDOl is frequently overexpressed in the solid tumor microenvironment, where its metabolic action of converting Tryptophan to Kynurenin, could interfere in the
proliferation of cytotoxic T-cells, expansion of Tregs and interference in memory T cell development (Lob et al. (2009) Nat. Rev. Cane. 9(6): 445-452; Zou (2005) Nat. Rev. Cane., 5(4): 263-274). In an attempt to overcome these immunosuppressive effects, a number of small molecule IDO inhibitors have been developed for cancer treatment, including IND (Hou et al. (2007) Cane. Res. 67(2): 792-801; Metz et al. (2012) Oncoimmunology, 1(9): 1460-1468). However, while IND has now been used in several clinical trials (including PDAC), its utility as a stand-alone immunostimulatory agent appears to be modest and the drug is therefore frequently combined with additional treatment modalities such as chemotherapeutics and checkpoint inhibitors (Soliman et al. (2014) Oncotarget, 5(18): 8136- 8146; Vacchelli et al. (2014) Oncoimmunology, 3(10): e957994). Oral administration of IND requires a high dose (up to 1200 mg BID) (McCormick et al. (2016) Hum. Vacc.
Immunother., 12(3): 563-575) to compensate for its poor water solubility, rapid blood clearance and limited accumulation at the tumor site (Soliman et al. (2016) Oncotarget, 7(16): 2292822938). These potentially unfavorable pharmacokinetics (PK) in humans was also corroborated by our animal data, which demonstrated that IV administration of IND is accompanied by a short circulatory half-life (t1/2) of <0.083 h, with < 0.1% of the injected dose gaining access to the tumor site (Figure 34, panel i). [0805] We hypothesized that the biodistribution and retention of IND can be improved at the PDAC site by a suitably designed nanocarrier. This was accomplished by using a labile ester bond to conjugate 1-methyl-D-tiyptophan to a single-chain phospholipid, l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PL) (Figure 26, panel a). The conjugation reaction for the design of this program was accomplished in 3 facile steps, namely: (i) protection of the IND amine group by Boc, (ii) esterification of Boc-IND with the PL, and (iii) Boc removal (Figure 26, panel a). The detailed synthesis and characterization of the intermediary components are described in Figure 34. The successful synthesis of IND-PL was confirmed by ESI-MS (C36H62N308P: [M+H]+, showing a calculated m/z of 696.4353 vs. the experimental measurment of 696.4379), 1H-NMR and 13C-NMR (Supplementary Figure 34, panels f, g). When suspended in aqueous media, the amphiphilic IND-PL could self- assemble into spherical 80 nm nanovesicles (IND-NV), as demonstrated by cryo-electron microscopy (cryoEM) (Figure 26, panel b, Figure 34, panel h). A UPLC-MS/MS procedure was used to study IND uptake and release in KPC cells exposed to IND-NV for different durations of time (Figure 35, panels a, b). Compared to free IND, the total intracellular IND content increased 35, 42.4, and 51.2-fold after exposure to IND-NV for 4, 24 and 72 h, respectively (Figure 26, panel c). We could also demonstrate that the uptake was
accompanied by increased IND released from the prodrug, yielding increases of 2.2, 9.9, and 22.2-fold, respectively, above the free drug at similar time points (Figure 26, panel c). An abiotic study confirmed that esterase activity and acidification of the incubation medium can release the IND from the prodrug; similar to what may happen in a cell (Figure 35, panel c).
[0806] IDO-mediated Trp depletion leads to triggering of immunosuppressive pathways, which may be impacted by the increased bioavailability of IND-PL in vitro and in vivo (Figure 26, panel d). IDO exerts an inhibitory effect on the mammalian target of rapamycin (mTOR) pathway, which controls the phosphorylation and activation of P-S6 kinase (Metz et al. (2012) Oncoimmunology, 1(9): 1460-1468). In addition, increased Kyn accumulation in the tumor site, allows IDO to induce aryl hydrocarbon receptor (AHR) expression as well as stimulating IL-6 production under control of the transcriptional factor, general control non-derepressible 2 (GNC2) (Figure 26, panel d) (Id.). Compared to free IND, treatment of KPC cells with IND-NV significantly interfered in these metabolic pathways, leading instead to increased P-S6K activation and downregulation of AHR and IL- 6 expression (Figure 26, panels e, f). This predicts that the increased potency of the conjugated drug in vitro may also translate into a more potent ability to reverse the immune suppressive effect of IDO in the TME, both for adaptive and innate immune responses (Hou et al. (2007) Cane. Res. 67(2): 792-801; Munn et al. (2004) J. Clin. Invest. 114(2): 280-290; Munn et al. (2002) Science, 297(5588): 1867-1870).
Co-administration of IND-NV with OX at the tumor site augments anti-PDAC immunity
[0807] While a vaccination provides proof-of-principle testing of the potential utility of ICD to initiate anti-PDAC immunity, there is no guarantee that de novo generation of an immune boosting response will be able to overcome the potent immunosuppressive environment in an already established tumor. We hypothesized that interference in the immunosuppressive PDAC environment may synergize with OX-induced ICD for an effective immune response. This idea was 1st tested in a SC tumor model to determine whether local injection of IND-NV could boost the OX-induced ICD effect (Figure 27). After allowing the KPC tumors to grow to 60-80 mm3 size, the mice received a single intratumoral (IT) injection of 1.25 mg/kg OX plus either a low (L) (2.5 mg/kg IND) or a high (H) (12.5 mg/kg IND) dose of IND-NV IT (Figure 27, panel a). Experimental controls included animals receiving IT injection with saline, free OX (1.25 mg/kg), free IND (12.5 mg/kg), or IND-NV (12.5 mg/kg IND) alone. Serial assessment of tumor volume, followed by animal sacrifice on day 31 for in situ inspection of the tumor size (Figure 27, panel c), demonstrated that OX plus IND-NV (H) treatment had the most robust tumor reduction effect, while OX plus IND-NV (L) or OX plus free IND (L or H) had lesser potency (Figure 27, panels b, c). Free IND had no effect on tumor growth, while IND-NV alone exerted a small effect (Figure 27, panels b, c).
[0808] The resected tumor tissues were used for IHC and multi-parameter flow cytometry analysis. IHC staining for CD8 and Foxp3 showed that OX plus IND-NV (H) resulted in significantly enhanced activation and recruitment of CD8 T cells along with a reduction in Foxp3+ T cells (Figure 27, panel d). Moreover, the comprehensive IHC profiles shown in Figures 36, panel a, and 30, panel b, demonstrate there was also good
responsiveness to OX alone, OX plus IND-NV (L), and OX plus IND (H or L), although not as prominent as OX plus IND-NV (H). These findings were corroborated by flow cytometry, demonstrating that the co-administration of OX plus IND-NV (H) significantly elevated the CD8+/Tregs ratio compared to other treatments, many of which (e.g., OX) differed significantly from saline and free IND (Figure 27, panel e). CD8 analysis was done by multiparameter staining for the CD45+/CD3+/CD8+/CD25+ population, while Tregs analysis involved staining for the CD45+/CD3+/CD4+/Foxp3+ population, as previously described (Pfirschke etal.(20\6) Immunity, 44(2): 343-354).
[0809] Since IDO also has a significant role in dendritic cells (DCs) participating in
ICD-enhanced tumor immunity, we asked whether IND co-administration could impact CD91 expression, which serves as the binding receptor on CRT+ DCs, as well as TLR4 that exerts an adjuvant effect upon interaction with FDVIGB-17 (Kroemer et al. (2013) Ann. Rev. Immunol, 31 : 51-72; Basu et a/. (2001) Immunity, 14(3): 303-313; Gardai et al. (2003) Cell, 115(1): 13-23). Multi-parameter flow cytometry showed that OX plus IND-NV (H) induced the most abundant CD91 expression in a CD45+/CD1 lb+/CDl lc+ cell population, compared to animals treated with free drugs, individually or combined (Figure 27, panel f). The phenotypic data was confirmed by IHC staining for CD91 in the tumor tissue (Figure 36, panel e). Moreover, IHC analysis of CRT and HMGB-1 expression demonstrated the most robust expression during treatment with OX plus IND-NV (H) (Figure 27, panel g). The comprehensive IHC profiles for CRT and HMGB-1 showed less robust responses to OX, OX plus IND (H and L) and OX plus IND-NV (L) (Figure 36, panels d, f). Likewise, assessment of TLR4 expression during IHC staining or multi-parameter flow cytometry analysis (of a CD45+/CD1 lb+/CDl lc+ APC population) confirmed the synergy between OX and IND-NV (H and L) (Supplementary Figure 36, panel g). Local OX plus IND-NVs (H) administration was also accompanied by significant IFN-γ release at the tumor site (likely from cytotoxic T cells), in parallel with increased training for activated (cleaved) caspase 3 (CC-3) (Figure 36, panels h, j). This was accomplished by decreased abundance of the anti-inflammatory cytokine, IL-10, which contributes to immune suppression in the TME (Figure 36, panel i). All considered, these data confirm that the combination of free OX plus IND-NV can induce synergistic anti-PDAC immunity, premised on activation of both the innate and adaptive immune systems.
Development of a dual delivery carrier for OX plus IND using a lipid-bilayer coated mesoporous silica nanoparticle (MSNP)
[0810] The 3r treatment protocol was established with a dual-delivery carrier to determine whether a combination of OX plus IND could act synergistically in an orthotopic KPC model, which closely mimics the growth and metastatic profile of human PDAC (Torres etal. (2013) PloSone, 8(11): e80580; Tseng et al. (2010) Clin. Cane. Res. 16(14): 3684-3695; Provenzano et al. (2013) Br. J. Cancer, 108(1): 1-8). The dual-carrier design was synthesized by coating the MSNP with a lipid bilayer that incorporates IND-PL, as well as serving to encapsulate OX in the porous interior (Figure 28, panel a). Careful
experimentation was undertaken to establish the optimal lipid bilayer composition. This was accomplished by using an IND-PL/Cholesterol/DSPE-PEG2K mixture at a molar ratio of 75:20:5 (Figure 37, panel a). The biofilm was laid down at the bottom of a round bottom flask, to which the OX-soaked MSNPs were added, followed by sonication (Liu et al. (2016) ACSNano, 10(2): 2702-2715). Details are described in the methods section. CryoEM images of the dual-delivery carrier showed particles of -100 nm size, uniformly coated with an intact bilayer, -5.5 nm thickness, slight-negative zeta potential and a loading capacity of 44.3 and 4.4 wt% for IND-PL and OX, respectively (Figure 28, panel a, Figure 37, panel b). These particles showed good colloidal stability in biological media for up to 30 days (Figure 37, panel b).
[0811] sualize the biodistribution of the IV-injected OX/IND-MSNP, 0.1% w/w
Dylight 680-labeled DMPE was incorporated into the lipid biofilm. This allowed IVIS imaging of the near-infrared (NIR) labeled particles at the orthotopic tumor site, as early as 2.5 h (Figure 28, panel b, upper panel). Particles were retained at the tumor site for up to 48 h, and could also be observed in the explanted organs, obtained from sacrificed animals (Figure 28, panel b, lower panel). The ex vivo images also showed biodistribution to the liver and spleen. The PK of OX and IND were assessed in a separate experiment, following IV injection of the OX/IND-MSNP carrier (5 mg/kg OX, 50 mg/kg IND) in tumor-bearing mice (Figure 28, panel c). Blood was collected at the indicated time points to measure OX and IND concentrations. Animals were sacrificed at 48 h to assess the IND content of the harvested organs by UPLC-MS/MS (Figure 28, panel d). Compared to free OX, MSNP encapsulation significantly prolonged the circulatory ti/2 of this drug from < 0.083 h to 10.4 h (Figure 28, panel c, left panel). The tm of IND was 9.5 h, which is significantly longer than the tl/2 < 0.083 h for free IND (Figure 28, panel c, lower panel, Figure 34, panel i).
Calculated as a % of the total injected dose, ~4 wt% of OX and IND could be seen to distribute to the tumor site by 48 h (Figure 28, panel d). These results were also confirmed by ICP-OES analysis of the tumor tissue to determine the content of elemental Si, which represent biodistribution of the carrier (Figure 38).
Dual delivery of OX plus IND-NV by MSNP induces effective anti-PDAC immunity in an orthotopic tumor model
[0812] Following orthotopic implantation of luciferase-expressing KPC cells in the pancreas of B6/129 mice, the animals were IV injected with the OX/IND-MSNP carrier on days 10, 14, 18, and 22 (Figure 29, panel a). Each animal received a MSNP particle dose of 111 mg/kg (equivalent 5 mg/kg OX and 50 mg/kg IND per injection). This drug ratio was premised on the IT dose calculations (Figure 27, panel c). The controls included mice receiving IV injection of saline, free OX, OX-MSNP (without IND-PL), IND-NV only, or IND-NV plus free OX at equivalent drug and/or particle doses. Monitoring of the orthotopic tumor growth by IVIS imaging was carried out at the indicated time points, with calculation of luminescence activity in the region of interest (ROI) (Figure 29, panel b). This demonstrated significantly higher rates of tumor shrinkage for the OX/IND-MSNP treated animals compared to the controls, including the OX-MSNP. Following animal sacrifice, autopsies were performed to allow ex vivo IVIS imaging of the primary tumor site, surrounding organs and sites of metastasis (Figure 29, panel c). The imaging data confirmed the highly significant reduction in the primary tumor size and metastatic spread during treatment with dual-delivery particles and OX-MSNP. The collective data set obtained for ex vivo imaging, is summarized in the heat map display in Figure 39, panels a, b. The rest of the controls showed large primary tumors and numerous metastatic foci. Kaplan-Meier plots to express animal survival, confirmed that while OX-MSNP significantly prolonged survival, the dual-delivery particles led to a significant additional survival improvement (Figure 29, panel d). The rest of the controls showed less favorable outcome. The imaging data were also confirmed by visual inspection of the organs during autopsy (Figure 29, panel c).
Measurement of serum amylase was used as a marker for pancreas invasion by the growing tumors44; this showed that the dual-delivery carrier and to lesser extent OX-MSNP, could decrease the serum enzyme levels, likely as a result of inhibiting tumor growth (Figure 29, panel e). [0813] Additional assessment of innate and adaptive immune features, as shown in the SC experiment (Figure 27), was carried out using IHC staining and multi-parameter flow cytometry. Assessment of CD8, Foxp-3, CRT, CD91, HMGB1, TLR4, IFN-γ, perforin, IL- 10 and CC-3 expression confirmed the observations of the direct tumor injection model (Figure 29, panel f, Figure 40, panels a-j). This confirms that the dual-delivery carrier and to some extent the OX-MSNP carrier, were capable of inducing a cytotoxic T cell response at the tumor site, disappearance of Tregs and induction of innate immunity, as described in Figure 27. In addition, we observed that the integrin marker, CD 103, was markedly upregulated in the CD45+/CD1 lb+/CDl lc+ cell population by OX and IND co-delivery (Figure 40, panel k). CD 103 -expressing DCs are particularly adapted for instruction of CD8+ T cells development and antitumor immunity (Broz et al. (2014) Cancer cell, 26(5): 638-652; Spranger et al. (2015) Nature, 523(7559): 231-235).
[0814] Based on the critical role of CD8-mediated cytotoxicity and the role of CD91 and TLR-4 in innate immunity (Figs. 27, 36), we asked, as an extension of the results in Figure 29, panel b, whether IV injection of antibodies to CD8 and TLR-4[40] or an injectable pool of siRNAs targeting CD91 (Pawaria & Binder (2011) Nat. Comm., 2: 521), could interfere with the protective immune response observed in this experiment {Id.) (Figure 41). Notably, these treatments had a significant inhibitory effect on the ability of OX/IND-MSNP to shrink tumor growth, provide prolonged survival, or ability to increase the CD87Tregs ratio (Figure 41, panels a-d). This was also reflected by IHC staining and the flow cytometry results, which showed a decline in the adaptive and innate immune responses (Figure 40, panels a-k).
[0815] The dual-delivery particle was well-tolerated in animal safety studies, without evidence of weight loss, increased liver enzymes (Figure 42) and interference in normal organ histology (data not shown). In contrast, free OX increased AST, ALT, and ALP levels.
[0816] To validate the involvement of the IDO metabolic pathway in the antitumor response, harvested tumor tissue was used for RT-PCR analysis of the expression of P-S6K, AHR, and IL-6 mRNA. We found that P-S6K was significantly upregulated with decreased AHR and IL-6 levels in the tumors of OX/IND-MSNP treated animals (Figure 29, panel g). These data agree with the in vitro results (Figure 26, panels e, f). Immuno-PET imaging confirms the generation of a systemic CD8+ T cell response in live animals treated with OX IND-MSNP
[0817] Immuno-positron emission tomography (immuno-PET) has been extensively employed in both pre-clinical and clinical studies to noninvasively and quantitatively track the presence and abundance of CD8+ T cells and other immune cell subsets following immunotherapies (Kim et al. (2016) Proc. Natl. Acad. Sci. USA, 113(15): 4027-4032; Radu et al. (2008) Nat. Med., 14(7): 783-788; Tavare et al. (2016) Cane. Res. 76(1): 73-82). This technique is potentially useful to determine the success of immunomodulatory therapy early on before the treatment impact on the tumor site can be assessed. To validate the tumor- infiltration and systemic activation of CD8+ T cells triggered by IV administration of
OX/IND-MSNP, a previous established 89Zr-desferrioxamine-labeled anti-CD8 cys-diabody (89Zr-malDFO-169 cDb) was used for immuno-PET monitoring of CD8+ T cells. This PET probe has high specificity for tracking newly-induced CD8+ T cell responses, as previously demonstrated by us (Tavare et al. (2016) Cane. Res. 76(1): 73-82; Tavare et al. (2015) J. Nucl. Med., 56(8): 1258-1264). We asked, therefore, whether PET imaging could be used to view the induction of an effective anti-PDAC immune response in live animals IV injected with saline, OX-MSNP (5 mg/kg OX) or OX/IND-MSNP (5 mg/kg OX and 50 mg/kg IND). Treatment was administered to the animals (n=3) on days 10, 14, 18, and 22 post-orthotopic implantation of KPC cells into the pancreas. The 89Zr-malDFO-169 cDb probe (29-63 μθ) was IV injected on day 26 and microPET and CT scans were obtained, using a G8 PET/CT scanner (Sofie Biosciences) at 20 h. Coronal (Figure 30, panel a, left panel) and transverse (Figure 30, panel a, right panel) view signal analysis for the localization of CD8+ T cells were obtained by AMIDE software.
[0818] Immuno-PET analysis showed background levels of CD8+ T cells appearing in a peripheral distribution in the tumors of saline-treated animals. This was accompanied by faint signals in the spleen and tumor draining lymph node (TDLN) (Figure 30, panel a, right panel). Since 89Zr-malDFO-169 cDb is eliminated renally, the kidneys showed intense levels of radioactivity (Tavare et al. (2016) Cane. Res. 76(1): 73-82). OX-MSNP treatment was associated with a modest increase in the overall radioactivity at the tumor sites, amounting to 2.5 -and 3.1 -fold increases, respectively, in the interior and peripheral tumor tissues (Figure 30, panel b). This increase likely reflects new infiltrating CD8+ T cells generated during the ICD response. This response was also accompanied by increased radioactivity in the spleen and tumor-draining lymph nodes (TDLN) (Figure 30, panel a, Figure 43). In contrast, treatment with OX/IND-MS P was accompanied by a prominent increase in the signal intensity in both the peripheral (7.5-fold) and interior (6.2-fold) tumor regions compared to saline. This is in keeping with the synergistic effect of IND-PL with the OX-induced ICD response (Figure 29). Moreover, there was a remarkable increase in signal intensity in the spleen and TDLN, as the hallmark of a systemic immune response. All considered, immuno- PET confirms the generation of an effective systemic immune response to PDAC in live animals, further boosting the IHC and flow cytometry data analysis, depicted in Figure 29.
Discussion
[0819] PDAC is an often-fatal and notoriously treatment-resistant disease, in desperate need of new treatment approaches for dealing with the primary tumor growth as well as metastatic spread. We demonstrate three treatment modalities to generate an anti- PDAC response, premised on the ability of OX to induce ICD. ICD is responsible for enhanced tumor antigen presentation as well as providing stimulatory effects to the participating DCs. This triggers the activation of cytotoxic T cells and anti-PDAC immunity that was synergistically enhanced by an intervention in the IDO pathway. A subcutaneous vaccination approach, which utilizes ex vivo induction of ICD by OX in a KPC cell line, it is suffice to a generate systemic immune response that can interfere with tumor growth at a remote site as well as allowing adoptive transfer to non-immune animals. The 2nd treatment modality involved local injection of OX plus an IND-PL nanovesicle to induce the recruitment of cytotoxic CD8+ lymphocytes, depletion of Tregs, reversal of the CD8+/Foxp3+ ratio, cytotoxic tumor killing, and tumor shrinkage at the local injection site. These adaptive immune responses were accompanied by boosting of the innate immune system, as reflected by CRT and HMGB1 expression, as well as the activation of a DC population, particularly well-suited for generating cytotoxic T cell responses. The 3rd treatment approach combined OX and IND-PL into a single MSNP -based nanocarrier, which allows systemic
biodistribution and drug delivery to orthotopic KPC tumor sites. The dual-delivery approach achieved synergistic enhancement of adaptive and innate anti-PDAC immunity, leading to a significant improvement in animal survival.
[0820] Our proposed nano-enabled approach for boosting immunotherapy offers distinct advantages over current immunotherapy strategies for PDAC, including peptide and protein vaccines (e.g., mutant Kras, survivin, vascular endothelial growth factor receptor, gastrin and heat shock proteins) (Paniccia et al. (2015) Chinese J. Cane. Res., 27(4): 376- 391), whole-cell vaccination approaches {e.g., PDAC cell lines engineered to express GM- CSF)Li8J, dendritic cell vaccines (Koido et al. (2014) Clin. Cane. Res., 20(16): 4228-4239), microorganisms {e.g., expression of antigenic peptides by vaccinia virus or heat-killed Mycobacterium obuense) (Strug et al. (2008) J. Proteome Res., 7(7): 2703-2711) and immune checkpoint blockade {e.g., anti-CTLA-4 or anti-PDl or monoclonal antibodies) (McCormick et al. (2016) Hum. Vacc. Immunother., 12(3): 563-575). While most of these approaches rely on select antigens chosen from the large repertoire of potential immunogenic PDAC components, the reality is that there is a dynamic interplay between the tumor and the immune system, which could render the use of specific antigens redundant, including through the process of immune editing or the display of T cell antigen receptors (TCR) of sub-optimal affinity or on/off rates (Dunn et al. (2004) Ann. Rev. Immunol. 22: 329-360). In contrast, the use of ICD prepares the dying cancer cells for uptake and processing by local APCs, with the possibility that the full complement of mutant or neo-antigens can participate in dynamically fashion in T cell selection, allowing effective TCR proofreading for immune activation. This allows the cognitive immune system to adapt to an array of continuously evolving tumor antigens rather than restricting the immune response to selected antigens.
[0821] The idea that ICD could be advantageous to mounting an anti-PDAC immune response is reflected by studies employing the whole cell vaccine, Algenpantucel-L; this vaccine is comprised of two irradiated PDAC cells, genetically engineered to express the murine enzyme, a (1, 3)-galactosyltransferase (aGT) (McCormick et al. (2016) Hum. Vacc. Immunother., 12(3): 563-575). aGT is responsible for the synthesis of the aGal epitope, e.g., in normal gut flora. This immune challenge leads to a constitutive anti-aGal response in the human host, in the form of a high titer of aGT antibodies. Thus, vaccination with the aGal- expressing cell lines leads to the induction of These antibodies lead to a hyper-acute immune response upon vaccination with Algenpantucel-L. The death of these cell lines leads to CRT- mediated tumor cell uptake and processing by DCs, which also receive adjuvant input in subsequent phases of tumor cell death (Obeid etal. (2017) Nat. Med., 13(1): 54-61; Tesniere et al. (2010) Oncogene, 29(4): 482-491). Noteworthy, data from a phase II clinical trial, using the aGal vaccine, have demonstrated the ability to induce a high titer of anti-CRT antibodies, which correlates with increased survival in PDAC patients (Rossi et al. (2014) J. Clin. Oncol. 32(5s): Suppl: abstr 3029).
[0822] Instead of using genetically engineered PDAC cells, we propose that ICD induction by a an already FDA-approved chemotherapeutic agent (such as OX) constitutes a more effective means to achieve anti-PDAC immunity because it targets autologous cancer cells rather than preselected PC cell lines (which may not dynamically display the full complement of tumor antigens). We also propose that it may be easier to adjust the dosimetry of chemotherapy-induced ICD rather than relying on a hyper-acute immune response that may not always induce ICD. Good experimental data have recently been collected to show the feasibility of using chemotherapy to induce ICD in lung or colon carcinoma, with the ability to amplify these responses by immune checkpoint blockade (Ffirsc ke et al.(2016) Immunity, 44(2): 343-354; Rossi et al. (2014) J. Clin. Oncol. 32(5s): Suppl: abstr 3029). Moreover, for colon cancer it has been demonstrated that core-shell nanoparticles, comprised of an OX core and a photosensitizing pyrolipid conjugate in the shell, can synergize to deliver an ICD response, which may be useful for a vaccination approach or an abscopal effect (He et al. (2016) Nat. Comm. 7: 12499). [
[0823] This is the 1st report demonstrating the use an ICD approach in PDAC through the use of nanocarriers. Our study also introduces the novel principle of using a nanocarrier to simultaneously induce ICD and immunomodulation. OX is an integral component of the FOLFIRINOX regimen, and constitutes one of a short list of chemotherapeutics capable of inducing ICD, other than anthracyclines (Kepp et al. (2014) Oncatarget, 5(14): 5190-5191). The unique ability of these chemotherapeutics to induce ICD is dependent on their ability to initiate a sequence of events that differ from regular apoptosis. Integral to ICD, is triggering of ER stress, which leads to CRT expression at the pre-mortem stage (Id.). CRT expression serves as an "eat me" signal for antigen-presenting DCs, which also receive adjuvant signals at subsequent stages of ICD by the release of the nuclear protein, HMGBl, and ATP from the dying tumor cells (Obeid etal. (2017) Nat. Med, 13(1): 54-61; Kroemer et al. (2013) Ann. Rev. Immunol, 31 : 51-72). CRT and HMGB-1 interacts with CD91 and TLR4, respectively. The involvement of these innate immune receptors was confirmed by IHC and flow cytometry analysis demonstrated in Figure 27, panels f, g, Figure 36, panels d-g, and Figure 40, panels c-f. Moreover, boosting of innate immunity in the a CD45+/CD1 lb+/CDl lc+ cell population is directly linked to generating a cytotoxic T cell response, which is carried out by IFN-γ, and perforin producing CD8+ lymphocytes (Figure 29, panel f, Figure 36, panels a, h, and Figure 40, panels a, g, h). [0824] Immune activation in the PDAC microenvironment has to overcome a number of immune suppressive mechanisms, including the presence of CD4+/Foxp3+ Tregs, secretion of anti-inflammatory cytokines, expression of checkpoint inhibitors and overproduction of IDO. While our results indicate that OX alone is capable of increasing the CD8+/Foxp3+ ratio at local and systemic tumor sites, the co-administration of a PL-conjugated IDO inhibitor, IND-PL, significantly enhanced this integrative response parameter, which reflects the transition from an immune suppressive to an immune stimulatory TME. This synergy reflects the importance of the IDO metabolic effect in the TME, in much the same way as regional expression of this enzyme please an immune surveillance role in the placenta to protect the fetus. [25] The delivery of IND as a prodrug also impacts the innate immune system, as demonstrated by enhanced expression of CRT and HMGB 1 by the dual delivery carrier (Figure 36, panels d, f, and Figure 40, panels c, e). In this regard, it is interesting that IND is capable of promoting autophagy, which plays a key role in ATP release during ICD.[28]
[0825] IDO inhibitors are currently undergoing clinical trials in several cancer types, including breast, prostate, melanoma, brain and pancreas. [31] This includes the use of IND together with gemcitabine, nab-paclitaxel and anti-PDLl antibody. [31] A major advantage of our nanocarrier approach is the improvement of the PK and intratumoral accumulation of IND-PL. Free IND is relatively water insoluble and has unfavorable PK characteristics. In contrast, IND-NV significantly increases the uptake and release of IND in tumor cells (Figure 26, panel c), which translates to a more robust interference in IDO-mediated immune suppressive signaling pathways in vitro and in vivo (Figure 26, panels e, f, and Figure 29, panel g). In addition to improving the circulatory t\a and PK of IND, the dual delivery carrier also improved the PK of OX (Figure 28, panel c, Figure 34, panel i). The harmonized PK and contemporaneous delivery further contributes to the in vivo synergy of the OX/IND- MSNP at the tumor site. The results are also confirmed, in live animals, by immuno-PET imaging (Figure 30).
[0826] How can this discovery be practically implemented to provide PDAC immunotherapy in the clinic? Based on our animal studies, possible ways to improve immunotherapy in patients could include: (i) tumor cell harvesting from resected cancer tissues during surgery, with the possibility of developing a cell culture-based vaccine approach; (ii) local injection of OX and IND-PL into the tumor under remote guidance, during collection of biopsies or direct visualization during surgery; (iii) systemic
administration of one or a combination of treatment modalities, which may include the use of free drugs, IND-NV or the dual-delivery carrier. In addition, it is also possible to enhance treatment efficacy by nanomaterials that exhibit intrinsic nanoscale properties and functions that lead to sequential induction of ER stress, ICD, autophagy and the release of adjuvants. It is also possible to use nanocarriers to deliver other FDA-approved drugs (e.g., cardiac glycosides, GADD34/PP1 inhibitors, Ca2+-activated K-channel agonists, poly-I/C, etc.)[U] to achieve ICD, individually or in combination with chemotherapeutics or ICD-inducing nanoparticles. Another approach could be to combine chemotherapy and IND delivering nanoparticles with immune checkpoint blockers, irradiation, photodynamic therapy or cytotoxic viruses to achieve additional immune response enhancement. The same principles could also apply to the treatment of a host of other cancers.
[0827] In summary, we demonstrate that nano-enabled methods for the delivery of
OX and IND to the PDAC site, allows effective combination of ICD and interference in an immune suppressive metabolic pathway to establish effective anti-PDAC immune responses that involve both innate and cognitive immunity. The nano-enabled approach can also be reduced to clinical practice by using the a vaccination approach, local treatment and systemic administration. The same approach may also apply to other cancers.
Methods Synthesis of the IND-PL prodrug.
[0828] This procedure was carried out in 3 steps, the 1st of which was synthesis ofN-
Boc-IND. IND (200 mg), di-tert-butyl dicarbonate (Boc, 260 mg) and NaHC03 (230 mg) were dissolved in a mixture containing 10 mL tetrahydrofuran (THF) and 10 mL H20. The sample was stirred at 0 °C for 15 min and then at room temperature overnight. THF was removed by evaporation, followed by the addition of IN HC1 (10 mL). The solution was brought to pH=l by crystal precipitation, followed by suction filtration to purify the pale yellow solid. The yield was determined by subtraction analysis (Figure 34, panel a) and synthesis success confirmed by 1H-NMR, 14C-NMR and ESI-MS (positive mode), as described online. Subsequent synthesis of N-Boc-IND-PL was performed by dissolving 100 mg l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PL), 150 mg N-Boc-IND, 156.7 mg EDC, 97.3 mg DMAP, and 146 mg DIPEA in water-free dichloromethane (DCM, 20 mL), while stirring for 48 h. The resulting pale yellow solution was obtained by funnel separation (repeated 3 times, using water). The DCM solution was vacuum-dried and purified by silica-gel chromatography, using a mobile phase comprised of
ethanol: chloroform: water (4:6: 1, v/v/v). Analysis of the yield, and characterization of the product was performed by NMRs and ESI-MS, as described online (Figure 34). In the final step, the synthesis of IND-PL was carried out by stirring 58.6 mg N-Boc-IND-PL in a mixture of 1 mL trifluoroacetic acid and 1 mL DCM for 6 h at room temperature. The solvent was removed by rotatory evaporation and the residue was re-dissolved in 400 μL DCM, to which 25 mL diethyl ether was added dropwise, followed by centrifugation to retrieve the pale- yellow solid. The washing step was repeated thrice using diethyl ether. The final product was comprehensively characterized for its purity and composition by MRs and ESI-MS.
Self-assembly of IND-PL into nanovesicles (Ind-NV).
[0829] The self-assembly of IND-PL into IND-NV was carried out by a variation of a liposome synthesis procedure. Briefly, 5 mg of IND-PL was dissolved in chloroform in a 50 mL round bottom glass flask. The solvent was evaporated under a rotatory vacuum to form a thin film, which was dried further under vacuum overnight. The film was hydrated with 1 mL of PBS and sonicated for 1 h. To obtain size-controlled IND-NV assembly, the suspension was extruded 13 times through a Mini -Extruder (Avanti Polar Lipids), using a polycarbonate membrane with 100 nm pores (Avanti Polar Lipids) at 80 °C. IND-NV size and morphology were assessed by dynamic light scattering and cryoEM, respectively (Figure 26, panel b).
Synthesis of the OX IND-MSNP nanocarrier.
[0830] A delivery carrier was designed by trapping OX in the mesoporous interior of an 80 nm MSNP, using a lipid bilayer into which IND-PL was incorporated. In order to achieve dual drug loading, we made use of a previously described biofilm method that rapidly encapsulates the MSNP core during energy input.[4' 5] This was accomplished by adding the OX-soaked MSNP suspension on top of a lipid biofilm. For loading OX into the pores of MSNP, 30 mg of OX was dissolved in 30 mg MSNP water solution (3 mL) with stirring overnight. Then the mixture was centrifuged at 15000 rpm for 15 min 3 times to remove the unencapsulated OX and resuspended in 3 mL DI water. Biofilm was comprised of a 60 mg lipid mixture, which included IND-PL, cholesterol, and DSPE-PEG2K in the molar ratio of 75:20:5. This ratio was derived at by experimenting with different lipid mixtures (Figure 37, panel a). The lipids were dissolved in chloroform at a concentration of 20 mg/ml, and added to a round bottom flask. Following rotary vacuum evaporation of the chloroform, the OX-soaked MSNP suspension was added to the uniformly dispersed lipid biofilm, and then sonicated with a probe sonicator for 1 h, using a 15/15 second on/off working cycle at a power output of 32.5W. Then drug-loaded particles were washed 3 times by centrifugation at 15000 RPM for 15 min to remove free liposomes, and re-suspended in DI water, saline, or PBS, as indicated. The purified OX/IND-MSNPs were fully characterized for size, charge, loading capacity, morphology and endotoxin level using DLS, UPLC-MS/MS, ICP-OES, cryoEM and the Chromogenic LAL Assay, respectively. An optimal particle batch was comprised of particles with size around 100 nm, slightly negative charge and suspension stability of at least one month. Control particles were synthesized by entrapping OX only without the addition of I D-PL to the lipid bilayer (OX-MS P).
Particles were stored at 4°C prior to use in cellular and animal experiments.
Efficacy Assessment of OX IND-MSNP using the KPC-Derived Orthotopic Model.
[0831] We utilized a previously described NCI protocol4 to calculate the maximum tolerated dose (MTD) of IV-injected free and encapsulated OX; this was determined to be 8 and 12 mg/kg, respectively. The corresponding MTD value for IV-injected IND-NV was >500 mg/kg. Orthotopic tumor-bearing B6/129 mice were randomly assigned to 9 groups, each including seven animals. The mice were IV injected with OX/IND-MSNP to deliver a dose of 5 mg/kg OX and 50 mg/kg IND every 4 days from day 10 onwards. The controls included equivalent doses of free OX, OX-laden particles only, IND-NV, IND-NV + free OX, delivered at the same administration frequency. We also included saline as a negative control. Tumor burden was monitored by IVIS imaging from day 10 onwards, allowing the bioluminescence imaging intensity to be quantitatively expressed in the operator-defined ROI. The tumor tissue and major organs were harvested for quantification of ex vivo bioluminescence image intensity. On day 36, tumors were harvested for flow cytometry, IHC analysis and determination of mRNA expression (for P-S6K, AHR, and IL-6) by real-time PCR. We also withdrew blood for amylase measurement and blood chemistry analysis. In order to assess the adaptive and innate immune changes leading to effective anti-PDAC immunity during OX/IND-MSNP treatment, we also asked whether dosing with anti-CD8 and anti-TLR4 antibodies or knockdown of CD91 by siRNA could change the outcome (Figure 41). The experiment was repeated once, using the same treatment comparisons, to determine the effect on survival rate in each group (n=7). Kaplan-Meier plots were used to express animal survival data. Statistical Analysis.
[0832] Statistical analysis was carried out with the SPSS statistical package (version
23, SPSS). Data sets were analyzed using a two-tailed Student's t-test, and one- or two-way analysis of variance, followed by Tukey's HSD test for multiple comparisons. Comparison of Kaplan Meier survival curves was performed with the Log-rank Mantel-Cox test. Results were expressed as mean ± SEM of at least three independent experiments. Statistical significance thresholds were set at *p <0.05; **p <0.01; #p <0.001. [0833] A detailed description of the methods for cell culture, the use of mice, assessment of CRT expression, HMGB-1 release, a vaccination approach for inducing systemic immunity, adoptive splenocyte transfer to non-immune animals, IHC and flow cytometry analysis, cellular processing of IND-NV, impact of IND-NV on immunoregulatory signaling pathways, use of a SC tumor model for local injection of drugs and carriers, pharmocokinetics of OX and delivery in the orthotopic KPC model, are provided in the supporting information.
Supplemental Methods
Cell and mice.
T ¾T
[0834] A KPC cell line, derived from a spontaneous tumor in a transgenic Kras Ui^' Trp53"™'zri'7Pdx-l-Cre mouse, was used for the cellular studies and growing subcutaneous and orthotopic tumors in mice (Hingorani et al. (2005) Cancer Cell, 7(5): 469- 483). We also obtained a PANC-1 cell line from ATCC. Both cell lines were cultured in complete DMEM medium, containing 10% FBS, 100 U/mL penicillin, 100 μg/mL
streptomycin, and 2 mM L-glutamine. To visualize KPC tumor growth by IVIS
bioluminescence imaging, the KPC cells were stably transfected with a luciferase expressing lentiviral vector in the vector core facility at UCLA (Liu et al. (2016) ACS Nano, 10(2): 2702-2715). Female B6/129 mice (Jackson Laboratory, 8-10 weeks old) were used to grow subcutaneous or orthotopic KPC tumors. The animals were maintained under pathogen-free conditions and all animal experiments were approved by the UCLA Animal Research Committee.
Assessment of CRT expression and HMGB-1 release from the cell lines.
[0835] 1 105 KPC or PANC-1 cells were seeded in 24-well plates overnight. The cell culture medium was removed and replenished with Cis, OX and DOX containing media at the indicated concentrations for 4 or 24 h. Supernatants were collected for HMGB-1 detection by an ELISA kit (IBL International GmbH), according to the manufacturer's instructions. To assess CRT expression by flow cytometry, cells were trypzinized, washed in cold PBS and then sequentially stained with a primary rabbit anti-CRT antibody (Ab2907, Abeam), followed by an Alexa Fluor®680-conjugated goat-anti rabbit IgG antibody for 30 min at 4 °C. The cells were incubated in 500 μΐ. PBS containing 50 μg/mL propodium iodide before washing and assessment in a LSRII flow cytometer (BD Biosciences). Data were expressed as fold-increase in mean fluorescence intensity (MFI) compared to the PBS control. The analysis was repeated once. Visualization of CRT expression was performed in KPC cells added to 8-well chamber slides (LAB-TEK®). Each well contained 1 * 104 KPC cells in 0.4 mL of culture medium. After incubation with 50 μΜ Cis, 50 μΜ OX, and 1 μΜ DOX for 4 h, cells were fixed and washed 3 times. Cells were stained with an ALEXA FLUOR® 647- conjugated anti-CRT antibody (abl96159, Abeam) for 30 min, followed by co-staining with 5 μg/mL ALEXA FLUOR® 488-conjugated wheat germ agglutinin (WGA) to visualize the cell surface membrane. Slides were mounted with Hoechst 33342 nuclear dye and visualized under a Leica SP8-SMD confocal microscope. High magnification images were obtained under the 63 χ objective lens. Vaccination approach to induce systemic immunity.
[0836] The timeline for the vaccination schedule is described in Figure 25, panel c.
KPC cells were exposed to PBS, 100 μΜ Cis, 50 μΜ OX and 1 μΜ DOX for 24 h to induce CRT expression. After confirmation of CRT expression by flow cytometry, 1 x 106 dying cells were injected twice into the right flank of B16/129 mice (n=7), 7 days apart. The same animals, 14 days after the 1st injection, received SC injection of viable KPC cell suspensions (1 x 106 cell in 0.1 mL DMEM/matrigel, 1/1, v/v) in the contralateral (left) flank. Tumor size was measured by a digital caliper every 3-4 days, and the volume calculated according to the formula π/6 x length x width (Liu et al. (2016) ACS Nano, 10(2): 2702-2715). Tumor burden was also monitored by IVIS imaging on day 7, 18, 25, and 29 and quantitatively expressed as luminescence signal intensity in the region of interest (ROI). The data were present as "spaghetti plots" that display the tumor growth in each individual animal. Statistical comparison of the groups was performed using two-way analysis of variance (SPSS).
Animals were sacrificed on day 29 and the tumors were harvested for flow cytometry and IHC analysis as described below. Demonstration of systemic immunity in surviving immune mice by attempted orthotopic tumor implant and adoptive transferred to non-immune animals.
[0837] The vaccination study described above produced 3 tumor-free survivors in the
OX group and 2 in the DOX group. These animals were used for a secondary tumor challenge by orthotopic pancreatic implant on day 74. This was accomplished by injecting 1 x 106 live KPC-luc cells into the tail of the pancreas after minor surgery, as previously described by us (Liu et al. (2016) ACS Nano, 10(2): 2702-2715). Tumor development was monitored by IVIS imaging. While the animals in the DOX-treated group developed pancreatic tumors, the animals in the OX-treated group remained tumor free. After sacrifice of previous Dox-treated survivors and harvesting of their splenocytes on day 132, adoptive transfer was performed to non-immune B 16/129 recipients (n=6). This was accomplished by injecting 3 >< 106 splenocytes IV. The controls consisted of 6 non-immunized animals injected with splenocytes from non-immune animals or 6 animals injected with splenocytes from saline-treated animals. Two days later, each of the groups was challenged by injection of 2x 105 KPC cells SC. To confirm the tumor specificity, 3 identical injected animal groups were used for challenge with B 16 melanoma cells injected SC.
Immunohistochemistry analysis.
[0838] In order to visualize the phenotypic changes during the induction of innate and cognate immune response through the use of the vaccination approach, local tumor injection or IV administration in orthotopic tumor bearing mice, IHC analysis was performed. Tumors harvested from the sacrificed animals were evenly divided into two parts, one for IHC and the other for flow cytometry. To prepare the tumor samples for IHC staining, the tumor pieces were fixed in 10% formalin followed by paraffin embedding. Tumor sections of 4 μπι thickness were mounted on glass slides by the UCLA Jonsson Comprehensive Cancer Center Translational Pathology Core Laboratory (TPCL) for hematoxylin-eosin (H&E) staining as well as a series of IHC staining procedures, following standardized protocols. Briefly, the slides were deparaffinized, incubated in 3% methanol-hydrogen peroxide, followed by 10 mM EDTA (pH=8) or 1 mM sodium citrate (pH=6) at 95°C using the Decloaking NxGen Chamber (Biocare Medical, DC2012). The slides were brought to room temperature, rinsed in PBST (Phosphate Buffered Saline containing 0.05% Tween-20) and then incubated with individual primary antibodies for 1 hour. The slides were rinsed with PBST and then incubated with appropriate HRP-conjugated secondary antibodies at room temperature for 30 minutes. After a rinse with PBST, the slides were incubated with DAB (3,3'- Diaminobenzidine) or Vulcan Fast Red Chromogen Kit 2 (for the CRT and CD91/LRP1 protocols only) (Biocare Medical, FR805) for visualization. Subsequently the slides were washed in tap water, counterstained with Harris' Hematoxylin, dehydrated in ethanol, and mounted with media. The slides were scanned by an Aperio AT Turbo Digital Pathology Scanner (Leica Biosystems) and interpreted by an experienced veterinary pathologist.
Antibody sources used for IHC.
[0839] Primary antibody sources and dilutions (2% BSA) obtained from Abeam included: anti-CD4 (abl83685, 1/200), anti-CRT (ab2907, 1/50), anti-HMGB-1 (abl8256, 1/200), anti-LRPl(CD91) (ab92544, 1/50), anti-TLR4 (abl3867, 1/50), and anti-perforin (abl6074, 1/100). Anti-CD8 (14-0808, 1/100), anti-Foxp3 (13-5773, 1/200) and anti-IL-10 (14-7101, 1/50) were from eBioscience. Anti-cleaved caspase 3 antibody was from Cell Signaling (#9664, 1/200) and anti-IFN-gamma from Novus Biologicals (NBP 1-19761, 1/200). Secondary antibodies included MACH2 Rabbit HRP-Polymer (Biocare Medical, RHRP520L) for IL-10 and TLR4; MACH2 Rabbit AP-Polymer (Biocare Medical,
RALP525) for CD91; Dako EnVision+ System HRP-labelled polymer Anti -Rabbit (Dako, K4003) for the remaining biomarkers.
Flow cytometry analysis.
[0840] The tumor pieces obtained for single cell analysis were cut into smaller pieces with scissors and digested in DMEM with 0.5 mg/mL collagenase type I (Worthington Biochemical Corporation) at 37 °C for 1 h. The digested tissues were gently meshed though a 70 μΜ cell strainer, twice. Red blood cells were lysed by Ack lysing buffer (Gibco) according to the manufacturer's instructions. The single-cell suspensions were washed twice and resuspended in staining buffer. Following cell counting and aliquoting, the suspensions were incubated with FcBlock (TruStain fcX™ anti-mouse CD16/32, clone 93, BioLegend) for 20 min to avoid nonspecific binding. Staining was then performed by using various combinations of fluorophore-conjugated antibodies for 40 min at 4 °C. The following anti- mouse antibodies were purchased from BD Biosciences: CD45-V450 (clone 30-F11), CD4- Alexa Fluor488 (clone RM4-5), Foxp3-PE (clone R16-715), CD8a-PE (clone 53-6.7), CDl lb-PE (clone Ml/70), CDl lc-V450 (clone HL3), CD284 (TLR4)-APC (clone SA15-21). CD103-Alexa Fluor 647 (clone 2E7) was purchased from BioLegend. LRP1 (CD91)-Alexa fluor 647 (clone EPR3724) was obtained from Abeam. CD3-APC-eFluor780 (clone 17A2) and CD25-APC (clone PC61.5) were purchased from eBiosciences. Multi-parameter staining was used to identify the following populations of interest: (i) CD8+ T cells
(CD45+CD3+CD8+CD25+), (ii) Tregs (CD45+CD3+CD4+Foxp3+), (iii) CD91+ DCs
(CD45+CD1 lb+CDl l+cCD91+), (iv) TLR4+ DCs (CD45+CD1 lb+CDl l+cTLR4+), and (v) CD103+ DCs (CD45+CD1 lb+CDl l+cCD103+). After washing, cells were used for flow cytometry analysis (brand name: LSRII, BS Biosciences). The data were processed by FlowJo software (Tree Star). Dead cells and doublets were excluded based on forward and side scatter.
Cellular uptake of IND-NV.
[0841] KPC cells were treated with free IND or IND-NV at the IND dose-equivalent of 100 μg/mL for 4, 24, and 72 h, respectively. This dosing is based on literature (Hou et al. (2007) Cancer Res. 67(2): 792-801). The cells were detached by trypsinization and extracted overnight in methanol to determine drug content by UPLC-MS/MS analysis. These extracts were added to a C18 Column (13θΑ, 1.7 μπι, 2.1 mm x 50 mm), connected to Waters LCT Premier with ACQUITY UPLC and Auto sampler. The separation condition involved gradient elution, 10 min per sample. The optimized separation procedure proceeded as follows: (i) 0 - 4.5 min, 95% water + 5% Acetonitrile; (ii) 4.5 - 6 min, 5% water + 95% Acetonitrile; and (iii) 6 -10 min, 95% water + 5% Acetonitrile. The flow rate was 0.4
Assessment of the impact of IND-NV on immunoregulatory signaling pathways in KPC cells.
[0842] 1 x 106 KPC cells were seeded into each well of a 6-well plate overnight, using tryptophan-deficient DMEM (Gibco) (Metz et al. (2012) Oncoimmunology, 1(9): 1460- 1468). After attachment, the cells were treated with IND or IND-NV at the indicated concentrations for 3 h. The supernatants were collected to assess IL-6 levels by an ELISA kit (BD Biosciences) according to the manufacturer's instructions. In order to determine the abundance of P-S6, AHR and actin by western blotting, cells were extracted in a lysis buffer, followed by electrophoresis on a 4-12% SDS-PAGE gel (Invitrogen, Grand Island, NY). The proteins were subsequently transferred to a PVDF membrane. After blocking in 5% BSA, the membrane was sequentially overlaid with primary and secondary antibodies and the blots developed by the addition of the ECL solution. The band intensity on the film was quantified by Image J software. Use of a SC tumor model for local injection of free OX plus IND-NV.
[0843] 1 106 KPC cells (in 100 μΐ, DMEM/matrigel, 1/1, v/v) were SC injected in the right flank of the animals. The dose design is based on literature (Tesniere et al. (2010) Oncogene, 29(4): 482-491). When tumors reached 60-80 mm3 in size, the mice received one- time IT administration of saline, free OX (1.25 mg/kg), free OX (1.25 mg/kg) + free IND (2.5 mg IND/kg), free OX (1.25 mg/kg) + free IND (12.5 mg/kg IND), free OX (1.25 mg/kg) + IND-NV (2.5 mg/kg IND) and free OX (1.25 mg/kg) + IND-NV (12.5 mg IND/kg). We also included a single IT injection of free IND (12.5 mg IND/kg) and IND-NV (12.5 mg IND/kg) as additional controls. Tumor burden was measured by a digital caliper, similar to what was described in the vaccination experiment. On day 31, tumors were harvested for flow cytometry and IHC analysis as described above.
Pharmocokinetics (PK) after IV injection of OX IND-MSNP in the orthotopic KPC model.
[0844] Orthotopic tumor bearing mice were used in this experiment (n=6). To visualize OX/IND-MSNP nanoparticle biodistribution in vivo, NIR-labeled OX/IND-MSNP was prepared by incorporating 0.1% w/w Dylight 680-labeled DMPE in the lipid biofilm (Liu et al. (2016) ACS Nano, 10(2): 2702-2715). For IVIS bioluminescence imaging of the tumor site, mice were injected intraperitoneally (IP) with 75 mg/kg D-Luciferin. Reference fluorescence images for the tumor-bearing mice were acquired prior to particle injection. Following a single IV injection of NIR-labeled OX/IND-MSNP, delivering the equivalent of 5 mg/kg OX and 50 mg/kg IND, mice were imaged after 0, 2.5, 8, 24, and 48 h. After sacrifice, ex vivo images were obtained for the harvested tumor, heart, liver, spleen, kidney, and lung tissues at 24 and 48 h. In a separate experiment, OX/IND-MSNP (5 mg/kg OX; 50 mg/kg IND) was IV administered to orthotopic KPC tumor bearing mice (n=6). Free OX served as a control. At the indicated time points, plasma was collected and digested in methanol or HNO3/H202 for UPLC-MS/MS (to measure IND&IND-PL) or to perform ICP-OES (for Si elemental analysis), respectively. The harvested tumor tissue and organs were also used to measure the drug and Si contents using by similar methods.
Immuno-PET imaging to confirm the systemic immune activation by OX IND- MSNP.
[0845] Immuno-PET imaging was performed following our previously established method (Tavare et al. (2016) Cancer Res. 76(1): 73-82; Tavare et al. (2015) J. Nuclear Med. 56(8): 1258-1264). Briefly, MalDFO-conjugated anti-CD8 cDb fragment was incubated for 1 h at room temperature at about 4-5 μθ 89Zr per μg protein. Radiolabeling efficiency was measured by ITLC (Biodex Medical Systems) using 20 mM citrate buffer pH 5.6 as the mobile phase. The ITLC strip was cut in half and sections were counted using a Wizard 3" 1480 Automatic Gamma Counter (Perkin-Elmer). Protein was purified using BioRad6 Spin columns equilibrated with PBS. Radiochemical purity was assessed by ITLC as above. 9 KPC orthotopic mice were established as described earlier. Saline, OX-MS P (5 mg OX/kg), and OX/IND-MS P (5 mg OX/kg and 50 mg IND/kg) were IV injected to mice (n=3) on day 10, 14, 18, and 22 for 4 consecutive administration post KPC tumor cells inoculation into pancreas. At day 26, 100 μΐ^ doses containing 1.07-2.33 MBq (29-63 μθ, 2.3-5.3 μCi/μg) 89Zr radiolabeled cDb in saline was IV injected to orthotopic KPC-tumor- bearing mice. 20 h later, mice were anesthetized and microPET and microCT scans were acquired using a G8 PET/CT scanner (Sofie Biosciences). MicroPET images were reconstructed by non-attenuation or scatter corrected maximum a posteriori (MAP) reconstruction. Images including coronal and transverse views were acquired and analyzed by AMIDE.
Table 9. Abbreviation List.
Figure imgf000195_0001
Example 6
Synthesis and Characterization of 1-L-MT-PL Prodrug [0846] Synthesis of a 1-L-MT-PL conjugate was similar to the 1-DT-MT-PL (a.k.a.
IND-PL) described herein (see also, Lu et al. (2017) Nat. Comm. 8: 1811). Briefly, three steps were involved:
[0847] 1) Protection of the amine group on 1-L-MT by coupling it with the Di-tert-butyl dicarbonate (Boc anhydride). 200 mg 1-L-MT was reacted with 260 mg Boc anhydride in the presence of 230 mg NaHC03 in 10 mL THF/10 mL H20 mixed solvent.
[0848] 2) Conjugating the OH- on PL to COOH- on 1-L-MT via esterification.
The resulting product, Boc protected 1-L-MT (150 mg), was conjugated to 100 mg single chain PL using mixed catalysts EDC (156.7 mg), DMAP (97.3 mg) and DIPEA (146 mg). The solvent was 20 mL dry DCM. The step 2 leads to Boc-l-L-MT-PL.
[0849] 3) Removal of the Boc group to render the active 1-L-MT-PL prodrug.
58.6 mg Boc- 1-L-MT was further treated using 1 mL TFA in 1 mL dry DCM solvent to remove Boc.
[0850] The final product, 1-L-MT-PL, was fully characterized by 1H-NMR, 13C-NMR and ESI-MS (see, Figure 50). The overall reaction yield is 28.2%. Our Mass spectrometry data confirmed the molecular weight of 1-L-MT-PL (i.e. 696.4368), which is very close to the calculated value of 696.4353.
[0851] An in vitro study was performed to demonstrate the cellular uptake and release of 1-L-MT-PL (see Figure 51). [0852] Western blot and ELISA were performed for P-S6K (cell lysate) and IL-6
(supernatant) in KPC cells treated with 1-L-MT-PL at 10 μΜ and 50 μΜ in tryptophan- deficient medium (see Figure 52).
[0853] Additionally, the effect of different isomers on in vitro IDO enzymatic assay in 4T1 breast cancer cells was determined (see Figure 53). [0854] The L-isomer is appears to be a direct inhibitor of IDO in our enzymatic activity assays. We also have cellular data that indicates in direct effects on the IDO pathway.
Example 7
Nanomaterial ICD Inducers
[0855] A number of immunogenic cell death (ICD) inducers are known to those of skill in the art. Illustrative ICD inducers include, but are not limited to oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, doxorubicin, epirubicin, idarubicin, mitoxanthrone, oxaliplatin, paclitaxel, R2016, and cyclophosphamide {see, e.g., Moon et al. (2015) J. ImmunoTherapy Cancer, 3 : 51; Bezu et al. (2015) Front. Immunol., 6: 187). Use of nanoparticles for the induction of immunogenic cell death.
[0856] The principles according to which various drugs listed are capable of inducing immunogenic cell death is the induction of an apoptosis-like cell death, which is
accompanied by an early cell stress response and effects on autophagy. This combination of cell stress with apoptosis (which is generally non-immunogenic), leads to a cell death process where there is an early expression of the cell stress response marker, calreticulum, which serves as an "eat me" signal for dendritic cells. This changes the response from non- immunogenic to immunogenic, further assisted by the release of HMGB l from the nucleus and ATP from the endoplasmic reticulum, which serves as immune adjuvants that stimulate the TLR4 and pure magic receptors, respectively. [0857] Through high throughput screening discovery aimed at understanding the hazard and safety of a vase number of nanomaterials in our nanomaterial safety laboratory, have taught us important lessons about nanomaterial physicochemical properties that can trigger cell death response pathways. These include nanomaterial properties {e.g., from transition metal oxides, rare earth oxides, graphene oxide) that induce oxidative stress, which can induce mitochondrial triggering and the initiation of apoptosis. Another example are rare earth oxide nanoparticles that can trigger a cell death response pathway by triggering lysosomal damage and interference in autophagy flux. These particles can induce cellular pyroptosis, which is a different form of inflammatory cell death. There are also nanoparticles such as fumed silica that could trigger cell death through disruption of the surface membrane. We used our nanomaterial libraries, to screen for materials that can induce immunogenic cell death, which can be assayed by following calreticulin (CRT) expression, HMGBl release etc.
[0858] Figurse 44A-44C show the results of screening of nanomaterials (NMs) {see, e.g., Table 10-for induced immunogenic cell death (ICD) in KPC pancreatic cancer cell after 24 h treatment with engineered nanoparticles. Calreticulin (CRT), one of the hallmarks dictating ICD, is translocated onto the cell surface membrane from endoplasmic reticulum following ICD inducer treatment. Flow cytometry analysis was performed to quantitatively measure the induction of CRT level compared to control group (Figure 44A). Figure 44B shows dose and time-dependent CRT induction in KPC cells. High mobility group box 1 protein (HMGB-1) is a second ICD signal, serving as an adjuvant to facilitate the innate immunity activation, which was released from the nucleus of the cells. ELISA was employed to detect the HMGB-1 concentration in the supernatant of the KPC cells after being treated with various Ms (Figure 44C). We demonstrated in vitro that CuO, Sb203, and GO-small are legitimate ICD inducers in cancer cells.
Table 10. Illustrative compounds screened for ICD-inducing activity.
Figure imgf000198_0001
AP-WMCNT 200 0.94
PF108- 200 1.13
MWCNT
COOH- 200 0.85
MWCNT
GO-S 200 6.10
GO-L 200 2.39
[0859] Figure 45 illustrates the cycoxicity profile of metal oxides (MOs), graphene oxides (GO), and carbon nanotubes (CNT) in KPC cells after 24 h treatment.
[0860] Figures 46A and 46B show the results of vaccination experiment using metal and metal oxide. Animal were treated using 2 rounds of vaccination (dying KPC cells treated with metal oxide nanoparticles) one week apart, followed by injecting live KPC cells SC on the contralateral side. Figure 46A shows spaghetti curves to show KPC tumor growth in the contralateral flank. IVIS imaging (Fig. 46B) was to monitor the tumor growth on the contralateral flank of mice shown in Fig. 46A. At the conclusion stage, the tumor tissues were used for flow cytometry experiment to measure CD8/Treg cell ratios (Fig. 46B).
[0861] Figures 47A-47C show the results of an intratumoral injection (IT) experiment using metal and metal oxide nanoparticle. KPC cells were subcutaneously injected into B6/129 mice. Figure 47A shows the results of a dose-seeking experiment for CuO
nanoparticle. The subQ tumors received single IT injection of CuO nanoparticle at 15, 30, 50 and 100 ug/mouse. The tumors were monitored up to 23 days. We used 50 ug CuO/mouse in the following IT experiment. In a pilot efficacy study using IT injection, KPC subQ tumor mice received single IT injection using indicated Ms (Figure 47B). The doses were shown in the figure. Tumor growth was monitored up to -23 days. At day 23, the tumors were harvested and single cell suspension was collected for flow analysis of various immune biomarkers. S ignificantly enhanced antitumor immunity was found in CuO group as confirmed by the boosted CD8/Treg ratio, granzyme B, IFN-gamma, etc. (Figure 47C). The IHC staining of tumor tissue is ongoing.
[0862] Figure 48, panels A-E, shows the results of an intratumoral injection (IT) experiment using GOs. The protocol is schematically illustrated in Figure 48, panel A. KPC cells were treated with GOs which induced ICD. The dying cells were used to vaccinate the mice and tumor size post-implantation was determined (Figure 48, panel B). IVIS imaging to monitor the KPC tumor growth on the contralateral flank of the mice as shown in Figure 48C. The CD8/Treg cell ratio was determined by flow cytometry (Figure 48, panel D). Selective IHC staining in the GO vaccination experiment, such as CD8, Fox-P3, CRT, Caspase 3 (CC3) and Perforin, are shown in Figure 48, panel E.
[0863] Figure 49 A and 49B show the results of an intratumoral injection (IT) experiment using GOs. KPC subQ tumor mice received single IT injection using indicated GOs (Figure 49A). The doses are shown in the figure. Tumor growth was monitored up to -23 days. At the conclusion stage, the tumor samples were harvested for CD8/Treg ratio measurement by flow cytometry (Fig. 49B). IP of anti-CD8 mAb (200 ug/mouse) interferes the ICD-mediated tumor inhibition induced by small GO (Fig. 49C).
[0864] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMS What is claimed is:
1. A nanovesicle drug carrier for the combined delivery of an IDO pathway inhibitor and an inducer of immunogenic cell death (ICD), said nanovesicle drug carrier comprising:
a lipid vesicle wherein said lipid vesicle comprises a lipid effective to form a vesicle comprising a lipid bilayer in an aqueous solution, and the lipid bilayer is associated with an inhibitor of the indoleamine 2,3 -di oxygenase (IDO) pathway (IDO pathway inhibitor); and
a cargo within said vesicle where said cargo comprises an agent that induces immunogenic cell death (ICD) (ICD-inducer).
2. The nanovesicle drug carrier of claim 1, wherein the IDO pathway inhibitor and the ICD inducer are synergistic in their activity against a cancer.
3. The nanovesicle drug carrier according to any one of claims 1-2, wherein said drug carrier, when administered systemically, delivers an amount of an ICD inducer effective to induce or to facilitate induction of immunogenic cell death of a cancer cell at a tumor site.
4. The nanovesicle drug carrier according to any one of claims 1-3, wherein said drug carrier, when administered systemically, delivers an amount of an IDO pathway inhibitor to partially or fully inhibit the IDO enzyme or IDO pathway at a cancer site.
5. The nanovesicle drug carrier according to any one of claims 1-4, wherein said IDO pathway inhibitor comprises an inhibitor of the IDO enzyme.
6. The nanovesicle drug carrier according to any one of claims 1-5, wherein said IDO pathway inhibitor comprises an inhibitor of the IDO pathway downstream from said IDO enzyme.
7. The nanovesicle drug carrier according to any one of claims 1-6, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l-methyl-tiyptophan (L-1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tryptophan (L-1MT), methylthiohydantoin-dl- tryptophan (MTH-Trp, Necrostatin), β-carbolines (e.g., 3-butyl-P-carboline),
Naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol- 3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S-methyl- dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl-brassinin, N- [2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S[(naphth-2- yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-3-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-4-yl)methyl]-dithiocarbamate, 5-bromo-brassinin, Phenylimidazole-based IDO inhibitors (e.g., 4-phenylimidazole), Exiguamine A, imidodicarbonimidic diamide,N-methyl-N'-9-phenanthrenyl-monohydrochloride
(NSC401366), INCB024360 (epacadostat), l-cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5- yl)ethanol (GDC-0919), IDO l -derived peptide, NLG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4- phenylimidazole.
8. The nanovesicle drug carrier of claim 7, wherein said IDO pathway inhibitor comprises 1-methyl-tiyptophan.
9. The nanovesicle drug carrier of claim 8, wherein said IDO pathway inhibitor comprises a "D" enantiomer of 1-methyl-tiyptophan (indoximod, 1-MT).
10. The nanovesicle drug carrier of claim 8, wherein said IDO pathway inhibitor comprises an "L" enantiomer of 1-methyl-tiyptophan (L-MT).
1 1. The nanovesicle drug carrier according to any one of claims 1-10, wherein said IDO pathway inhibitor, is disposed in a lipid comprising said vesicle and/or conjugated to a lipid comprising said vesicle.
12. The nanovesicle drug carrier according to any one of claims 1-10, wherein said vesicle comprises a phospholipid and/or a phospholipid prodrug.
13. The nanovesicle drug carrier of claim 12, wherein said vesicle comprises a phospholipid, and cholesterol (CHOL).
14. The nanovesicle drug carrier according to any one of claims 12-13, wherein said phospholipid comprises a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains.
15. The nanovesicle drug carrier of claim 14, wherein said phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), and diactylphosphatidylcholine (DAPC).
16. The nanovesicle drug carrier of claim 14, wherein said phospholipid comprises a natural lipid selected from the group consisting of egg phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC).
17. The nanovesicle drug carrier of claim 14, wherein said phospholipid comprises an unsaturated fatty acid selected from the group consisting of 1,2-dimyristoleoyl- sn-glycero-3 -phosphocholine, 1 ,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine, 1 ,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC), and l,2-dieicosenoyl-sn-glycero-3 -phosphocholine.
18. The nanovesicle drug carrier according to any one of claims 12-17, wherein said vesicle comprises an mPEG phospholipid with a phospholipid C14-C18 carbon chain, and a PEG molecular weight ranging from about 350 Da to 5000 Da.
19. The nanovesicle drug carrier of claim 18, wherein said vesicle comprises l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (DSPE-PEG).
20. The nanovesicle drug carrier of claim 19, wherein said vesicle comprises DPSE-PEG2K.
21. The nanovesicle drug carrier according to any one of claims 1-20, wherein said IDO pathway inhibitor is conjugated to a component of said vesicle.
22. The nanovesicle drug carrier of claim 21, wherein said IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid.
23. The nanovesicle drug carrier according to any one of claims 21-22, wherein said IDO pathway inhibitor is conjugated directly to said moiety.
24 The nanovesicle drug carrier according to any one of claims 21-22, wherein said IDO pathway inhibitor is conjugated to said moiety via a linker.
25 The nanovesicle drug carrier according to any one of claims 21-22, wherein said IDO pathway inhibitor is conjugated to PGHP.
26 The nanovesicle drug carrier according to any one of claims 21-24, wherein said IDO pathway inhibitor is conjugated to vitamin E.
27. The nanovesicle drug carrier according to any one of claims 21-24, wherein said IDO pathway inhibitor is conjugated to cholesterol (CHOL), or squalene.
28. The nanovesicle drug carrier according to any one of claims 21-24, wherein said IDO pathway inhibitor is conjugated to a fatty acid.
29. The nanovesicle drug carrier of claim 28, wherein said IDO pathway is conjugated to oleic acid or docosahexaenoic acid.
30. The nanovesicle drug carrier of claim 28, wherein said IDO pathway is conjugated to oleic acid or docosahexaenoic acid via an HO-(CH2)n=2-5-OH linker.
31. The nanovesicle drug carrier according to any one of claims 21 -24, wherein said IDO pathway inhibitor is conjugated to a lipid.
32. The nanovesicle drug carrier of claim 31, wherein said IDO pathway inhibitor is conjugated to a phospholipid comprising said lipid vesicle, said phospholipid thereby forming a phospholipid prodrug.
33. The nanovesicle drug carrier of claim 32, wherein said phospholipid prodrug comprises l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PL).
34. The nanovesicle drug carrier of claim 33, wherein said phospholipid prodrug comprises the structure:
Figure imgf000205_0001
35. The nanovesicle drug carrier of claim 34, wherein the 1-methyl- tryptophan component of said conjugated IDO pathway inhibitor comprises a "D" isomer methyl-tyrptophan (in e formula:
Figure imgf000205_0002
36. The nanovesicle drug carrier of claim 34, wherein the 1-methyl- tryptophan component of said conjugated IDO pathway inhibitor comprises an "L" isomer of 1 methyl -tyrptophan (L-1MT) characterized by the formula:
Figure imgf000205_0003
37. The nanovesicle drug carrier of claim 34, wherein the 1-methyl- tryptophan component of said conjugated IDO pathway inhibitor comprises a mixture of "D" and "L" isomers of 1-methyl-tiyptophan.
38. The nanovesicle drug carrier according to any one of claims 34-37, wherein said vesicle comprises IND-PL/Chol/DSPE-PEG.
39. The nanovesicle drug carrier of claim 38, wherein said vesicle comprises about 75% IND-PL, about 20% cholesterol, and about 5% DSPE-PEG2K.
40. The nanovesicle drug carrier according to any one of claims 1-39, wherein said ICD inducer comprises a chemotherapeutic agent selected from the group consisting of doxorubicin, oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, and cyclophosphamide.
41. The nanovesicle drug carrier of claim 40, wherein said ICD inducer comprises doxorubicin.
42. The nanovesicle drug carrier drug carrier according to any one of claims 1-41, wherein said carrier is colloidally stable.
43. The nanovesicle drug carrier according to any one of claims 1-42, wherein when the cargo in the nanocarrier is a weak base, said carrier comprises a cargo- trapping agent.
44. The nanovesicle drug carrier of claim 43, wherein said cargo trapping agent before reaction with the cargo drug loaded in the vesicle, is selected from the group consisting of triethylammonium sucrose octasulfate (TEA8SOS), ( IT^SC^, an ammonium salt, a trimethylammonium salt, and a triethylammonium salt.
45. The nanovesicle drug carrier of claim 44, wherein said cargo-trapping agent before reaction with said drug is ammonium sulfate.
46. The nanovesicle drug carrier according to any one of claims 1-45, wherein said drug carrier is conjugated to a moiety selected from the group consisting of a targeting moiety, a fusogenic peptide, and a transport peptide.
47. The nanovesicle drug carrier of claim 46, wherein said drug carrier is conjugated to a peptide that binds a receptor on a cancer cell or tumor blood vessel.
48. The nanovesicle drug carrier of claim 47, wherein said drug carrier is conjugated to an iRGD peptide.
49. The nanovesicle drug carrier of claim 47, wherein said drug carrier is conjugated to a targeting peptide shown in Table 5.
50. The nanovesicle drug carrier according to any one of claims 46-49, wherein said drug carrier is conjugated to transferrin, and/or ApoE, and/or folate.
51. The nanovesicle drug carrier according to any one of claims 46-50, wherein said drug carrier is conjugated to a targeting moiety that comprises an antibody that binds to a cancer marker.
52. The nanovesicle drug carrier of claim 51, wherein said drug carrier is conjugated to a targeting moiety that comprises an antibody that binds a cancer marker shown in Table 4.
53. The nanovesicle drug carrier according to any one of claims 51-52, wherein said antibody is selected from the group consisting of an intact immunoglobulin, an F(ab)'2, a Fab, a single chain antibody, a diabody, an affibody, a unibody, and a nanobody.
54. The nanovesicle drug carrier according to any one of claims 1-53, wherein said drug carriers in suspension are stable for at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months when stored at 4°C.
55. The nanovesicle drug carrier according to any one of claims 1-54, wherein said nanoparticle drug carrier forms a stable suspension on rehydration after lyophilization.
56. The nanovesicle drug carrier according to any one of claims 1-55, wherein said nanoparticle drug carriers, show reduced drug toxicity as compared to free drug.
57. The nanovesicle drug carrier according to any one of claims 1-56, wherein said nanoparticle drug carrier has colloidal stability in physiological fluids with pH 7.4 and remains monodisperse to allow systemic biodistribution and is capable of entering a disease site by vascular leakage (EPR effect) or transcytosis.
58. The nanovesicle drug carrier according to any one of claims 1-57, wherein said cargo within said vesicle comprises an agent that induces immunogenic cell death (ICD) selected from the group consisting of oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, doxorubicin, epirubicin, idarubicin, mitoxanthrone, paclitaxel, R2016, cyclophosphamide, and a bioreactive nanomatenal that induces ICD.
59. The nanovesicle drug canier of claim 58, wherein said cargo comprises oxaliplatin.
60. The nanovesicle drug canier of claim 58, wherein said cargo comprises doxorubicin.
61. The nanovesicle drug canier of claim 58, wherein said cargo comprises a bioreactive nanomaterial that induces ICD and/or innate immune activation.
62. The nanovesicle drug canier of claim 61, wherein said cargo comprises a nanomaterial that induces ICD where said nanomaterial is selected from the group consisting of CuO, Cu20, Sb203, As203, Bi203, P203, ZnO, Ti02, graphene oxide, and bioreactive 2D materials other than graphene or graphene oxide.
63. The nanovesicle drug carrier of claim 62, wherein said nanomaterial comprises copper oxide (e.g. CuO).
64. The nanovesicle drug carrier of claim 62, wherein said nanomaterial comprises Sb203.
65. A method of treating a cancer, said method comprising:
administering to a subject in need thereof an effective amount of a nanovesicle drug carrier according to any one of claims 1-64.
66. The method of claim 65, wherein the ICD inducer and the IDO inhibitor are synergistic in their activity against said cancer.
67. The method according to any one of claims 65-66, wherein said ICD- inducer is in an amount effective to elevate calreticulin (CRT) expression in cells of said cancer.
68. The method according to any one of claims 65-67, wherein said ICD- inducer is in an amount effective to elevate expression and/or release of HMGB 1 and/or induction of ATP release.
69. The method according to any one of claims 65-68, wherein said administering to a subject in need thereof an effective amount of a nanovesicle drug carrier comprises a primary therapy in a chemotherapeutic regimen.
70. The method according to any one of claims 65-68, wherein said administering to a subject in need thereof an effective amount of a nanovesicle drug carrier comprises an adjunct therapy in a treatment regime that additionally comprises chemotherapy using another chemotherapeutic agent, and/or surgical resection of a tumor mass, and/or radiotherapy.
71. The method according to any one of claims 65-70, wherein said nanoparticle drug carrier and/or said pharmaceutical formulation is a component in a multidrug chemotherapeutic regimen.
72. The method according to any one of claims 65-71, wherein said cancer is pancreatic ductal adenocarcinoma (PDAC).
73. The method according to any one of claims 65-71, wherein said cancer is a cancer selected from the group consisting of breast cancer, lung cancer, melanoma, pancreas cancer, liver cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi sarcoma, lymphoma), anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kaposi sarcoma, kidney cancer (e.g., renal cell, Wilm's tumor, and other kidney tumors), langerhans cell histiocytosis, laryngeal cancer, leukemia, acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myelogenous (CML), hairy cell, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., childhood, non-small cell, small cell), lymphoma (e.g., AIDS-related, Burkitt (e.g., non-Hodgkin lymphoma), cutaneous T-Cell (e.g., mycosis fungoides, Sezary syndrome), Hodgkin, non- Hodgkin, primary central nervous system (CNS)), macroglobulinemia, Waldenstrom, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma (e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, Myelogenous Leukemia, Chronic (CML), multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer , pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Ewing, Kaposi, osteosarcoma, rhadomyosarcoma, soft tissue, uterine), Sezary syndrome, skin cancer (e.g., melanoma, merkel cell carcinoma, basal cell carcinoma, nonmelanoma), small intestine cancer, squamous cell carcinoma, squamous neck cancer with occult primary, stomach (gastric) cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom
macroglobulinemia, and Wilm's tumor.
74. The method according to any one of claims 65-73, wherein said administration is via a route selected from the group consisting of intravenous administration, intraarterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation (including intranasal and intratracheal delivery, intracranial administration via a cannula, and subcutaneous or intramuscular depot deposition.
75. The method according to any one of claims 65-73, wherein said administration comprises systemic administration via injection or cannula.
76. The method according to any one of claims 65-73, wherein said administration is administration to an intra-tumoral or peri-tumoral site.
77. The method according to any one of claims 65-76, wherein said mammal is a human.
78. The method according to any one of claims 65-76, wherein said mammal is a non-human mammal.
79. The method according to any one of claims 65-78, wherein said cancer comprises PDAC, and said IDO pathway inhibitor comprises an agent selected from the group consisting of D-l-methyl-tiyptophan (indoximod, D-1MT), L-l-methyl-tiyptophan (L- 1MT), and a mixture of D-1MT and L-1MT, epacadostat.
80. The method of claim 79, wherein said inhibitor of an IDO pathway comprises D-l-methyl-tiyptophan (indoximod, D-1MT).
81. The method of claim 79, wherein said inhibitor of an IDO pathway comprises L-l-methyl-tiyptophan (L-1MT).
82. The method of claim 79, wherein said inhibitor of an IDO pathway comprises a mixture of D-1MT and L-1MT.
83. The method according to any one of claims 65-82, wherein said cargo within said vesicle comprises one oe more drugs selected from the group consisting of doxorubicin, paclitaxel, docetaxel, cyclophosphamide, mitroxantrone, etoposide, and bortezomib.
84. The method of claim 83, wherein said cargo within said vesicle comprises doxorubicin.
85. The method according to any one of claims 83-84, wherein said cancer is a breast cancer.
86. The method according to any one of claims 65-82, wherein said cargo within said vesicle comprises one or more drugs selected from the group consisting of oxaliplatin, gemcitabine, taxanes (paclitaxel and docetaxel), doxorubicin, etoposide.
87. The method of claim 86, wherein said cargo within said vesicle comprises oxaliplatin.
88. The method according to any one of claims 86-87, wherein said cancer comprise pancreatic cancer.
89. The method according to any one of claims 65-82, wherein said cargo within said vesicle comprises one or more drugs selected from the group consisting of a taxane (e.g., paclitaxel and docetaxel), gemcitabine, a Pt-based drug (e.g., cisplatin, oxaliplatin, carboplatin), cyclophosphamide, oxaliplatin plus cyclophosphamide, epirubicin (anthracycline), and etoposide.
90. The method of claim 89, wherein said cancer comprises lung cancer.
91. The method according to any one of claims 65-90, wherein said nanovesicle drug carrier is administered in conjunction with administration of an immune checkpoint inhibitor.
92. The method of claim 91, wherein said immune checkpoint inhibitor comprises an inhibitor of PD-1, PD-Ll, PD-L2, PD-L3, PD-L4, CTLA-4, LAG3, B7-H3, B7- H4, KIR and/or TIM3.
93. The method of claim 92, wherein said checkpoint inhibitor comprises an antibody that inhibits a moiety selected from the group consisting of PD-1, PD-Ll, and
CTLA4.
94 The method of claim 93, wherein said antibody comprises an antibody that inhibits PD-1
95. The method of claim 94, wherein said antibody comprises Pembrolizumab (Keytruda), or Nivolumab (Opdivo).
96. The method of claim 93, wherein said antibody comprises an antibody that inhibits PD-L1.
97. The method of claim 96, wherein said antibody comprises Atezolizumab (Tecentriq), Avelumab (Bavencio), or Durvalumab (Imfinzi).
98. The method of claim 93, wherein said antibody comprises an antibody that inhibits CTLA-4.
99. The method of claim 98, wherein said antibody comprises Ipilimumab
(Yervoy).
100. The method according to any one of claims 91-99, wherein the activity of said nanovesicle drug carrier and said immune checkpoint inhibitor is synergistic.
101. A composition comprising an IDO pathway inhibitor conjugated to a moiety that forms a nanovesicle in aqueous solution.
102. The composition of claim 101, wherein said IDO pathway inhibitor is conjugated to a moiety selected from the group consisting of a lipid, PHGP, vitamin E, cholesterol, and a fatty acid.
103. The composition according to any one of claims 101-102, wherein IDO pathway inhibitor is conjugated directly to said moiety.
104. The composition according to any one of claims 101-102, wherein IDO pathway inhibitor is conjugated to said moiety via a linker.
105. The composition of claim 104, wherein said linker comprises squalene.
106. The composition according to any one of claims 102-105, wherein said IDO pathway inhibitor is conjugated to PGHP.
107. The composition according to any one of claims 102-105, wherein said IDO pathway inhibitor is conjugated to vitamin E.
108. The composition according to any one of claims 102-105, wherein said
IDO pathway inhibitor is conjugated to cholesterol (CHOL).
109. The composition according to any one of claims 102-105, wherein said IDO pathway inhibitor is conjugated to a fatty acid.
110. The composition of claim 109, wherein said IDO pathway inhibitor is conjugated to oleic acid or docosahexaenoic acid.
111. The composition of claim 110, wherein said IDO pathway inhibitor is conjugated to oleic acid or docosahexaenoic acid via an HO-(CH2)n=2-5-OH linker.
112. The composition according to any one of claims 102-105, wherein said IDO pathway inhibitor is conjugated to a lipid.
113. The composition of claim 112, wherein said IDO pathway inhibitor is conjugated to a phospholipid.
114. The composition of claim 113, wherein said IDO pathway inhibitor is conjugated to a phospholipid comprising a saturated fatty acid with a C14-C20 carbon chain, and/or an unsaturated fatty acid with a C14-C20 carbon chain, and/or a natural lipid comprising a mixture of fatty acids with C12-C20 carbon chains.
115. The composition of claim 114, wherein said phospholipid comprises a saturated fatty acid selected from the group consisting of phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), and diactylphosphatidylcholine (DAPC).
116. The composition of claim 114, wherein said phospholipid comprises a natural lipid selected from the group consisting of egg phosphatidylcholine (egg PC), and soy phosphatidylcholine (soy PC).
117. The composition of claim 114, wherein said phospholipid comprises an unsaturated fatty acid selected from the group consisting of l,2-dimyristoleoyl-sn-glycero-3- phosphocholine, 1 ,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine, 1 ,2-dioleoyl-sn-glycero-3 - phosphocholine (DOPC), and l,2-dieicosenoyl-sn-glycero-3 -phosphocholine.
118. The composition of claim 114, wherein said phospholipid comprises 1- palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine.
1 19. The composition according to any one of claims 101-1 18, wherein said IDO pathway inhibitor comprises an agent selected from the group consisting of D-l-methyl- tryptophan (indoximod, D-1MT), L-l-methyl-tiyptophan (L-1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tiyptophan (L-1MT), methylthiohydantoin-dl -tryptophan (MTH-Trp, Necrostatin), β-carbolines (e.g., 3-butyl-P-carboline), Naphthoquinone-based (e.g., annulin- B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S- methyl-dithiocarbamate, S-hexyl-brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl- dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S[(naphth-2-yl)methyl]-dithiocarbamate, N-[2- (indol-3-yl)ethyl]-S-[(pyrid-3-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid- 4-yl)methyl]-dithiocarbamate, 5-bromo-brassinin, Phenylimidazole-based IDO inhibitors (e.g., 4-phenylimidazole), Exiguamine A, imidodicarbonimidic diamide,N-methyl-N'-9- phenanthrenyl-monohydrochloride (NSC401366), INCB024360 (Epacadostat), 1- cyclohexyl-2-(5H-imidazo[5, l-a]isoindol-5-yl)ethanol (GDC-0919), IDO 1 -derived peptide, LG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl- 2-thiopseudourea hydrochloride, and 4-phenylimidazole.
120. The composition according to any one of claims 101-1 18, wherein said IDO pathway inhibitor comprises 1 -methyl -tryptophan.
121. The composition of claim 120, wherein said IDO pathway inhibitor comprises a D isomer of 1-methyl-tiyptophpan.
122. The composition of claim 120, wherein said IDO pathway inhibitor comprises an L isomer of 1-methyl-tiyptophpan.
123. The composition of claim 120, wherein said IDO pathway inhibitor comprises a mixture of D and L isomers of 1-methyl-tiyptophpan.
124. The composition of claim 120, wherein the lipid conjugated IDO pathway inhibitor comprises l -palmitoyl -2-hydroxy-sn-glycero-3-phosphocholine.
125. The composition of claim 124, wherein the lipid conjugated IDO pathway inhibitor comprises a compound having the structure: IND-PL
126. The composition of claim 125, wherein the 1-methyl-tryptophan component of said conjugated IDO pathway inhibitor comprises a "D" isomer of 1 methyl- tyrptophan (indoximo a:
Figure imgf000216_0001
127. The composition of claim 125, wherein the 1-methyl-tryptophan component of said conjugated IDO pathway inhibitor comprises an "L" isomer of 1 methyl- tyrptophan (L-1MT) characterized by the formula:
Figure imgf000216_0002
128. The composition of claim 125, wherein the 1-methyl-tryptophan component of said conjugated IDO pathway inhibitor comprises a mixture of "D" and "L" isomers of 1-methyl-tryptophan.
129. The composition according to any one of claims 101-128, wherein the lipid-conjugated IDO pathway inhibitor forms a component of a vesicle.
130. A nanoparticle drug carrier for the combined delivery of an IDO pathway inhibitor and an inducer of immunogenic cell death (ICD), said nanoparticle drug carrier comprising:
a mesoporous silica nanoparticle having a surface and defining a plurality of pores that are suitable to receive molecules therein;
a lipid bilayer coating the surface;
a first cargo comprising an inhibitor of the indoleamine 2,3- dioxygenase (IDO) pathway (IDO pathway inhibitor); and
a second cargo comprising an agent that induces immunogenic cell death (ICD) (ICD-inducer);
wherein the lipid bilayer is substantially continuous and encapsulates said nanoparticle stably sealing the plurality of pores.
131. The nanoparticle drug carrier of claim 130, wherein said nanoparticle drug carrier contains a predefined ratio of IDO pathway inhibitor to ICD-inducer.
132. The nanoparticle drug carrier according to any one of claims 130-131, wherein the IDO pathway inhibitor and the ICD inducer are synergistic in their activity against a cancer.
133. A nanomaterial carrier for the combined delivery of an inhibitor of the IDO pathway and an inducer of immunogenic cell death (ICD), said nanomaterial carrier comprising:
a nanomaterial that induces ICD; and
a lipid or lipid formulation comprising an IDO pathway inhibitor where said lipid or lipid formulation is disposed on the surface of said nanomaterial.
134. The nanomaterial carrier of claim 133, wherein said lipid or lipid formulation fully encapsulates said nanomaterial.
135. The nanomaterial carrier according to any one of claims 133-134, wherein said lipid or lipid formulation is not a lipid bilayer.
136. The nanomaterial carrier according to any one of claims 133-134, wherein said lipid or lipid formulation comprises a lipid bilayer.
137. The nanomaterial carrier according to any one of claims 133-136, wherein said nanomaterial comprises a material selected from the group consisting of selected from the group consisting of CuO, Cu20, Sb203, AS2O3, B12O3, P2O3, ZnO, Ti02, graphene oxide, and 2D materials other than graphene or graphene oxide.
138. A pharmaceutical formulation said formulation comprising:
a plurality of nanovesicle drug carriers according to any one of claims 1-57, and/or a plurality of nanoparticle drug carriers according to any one of claims 130-132, and/or a plurality of nanomaterial carriers according to any one of claims 133-137; and
a pharmaceutically acceptable carrier.
139. A method of treating a cancer in a mammal, said method comprising: administering to an intra-tumoral or peri-tumoral site an effective amount of an IDO pathway inhibitor in conjunction with an effective amount of an agent that induces immunogenic cell death (ICD) (an ICD-inducer).
140. A method of treating a cancer, said method comprising:
administering to a subject in need thereof an effective amount of a nanoparticle drug carrier according to any one of claims 130-132; and/or
a nanomaterial carrier according to any one of claims 133-137.
141. A method for the treatment and/or prevention of a cancer in a mammal, said method comprising:
providing cancer cells in which immunogenic cell death (ICD) has been induced ex vivo; and
vaccinating said mammal with said cells, where said vaccination induces an anti-cancer immunogenic response.
142. A kit for the treatment or prophylaxis of a cancer said kit comprising: a container containing an IDO pathway inhibitor; and/or
a container containing an agent that induces immunogenic cell death (ICD) (ICD-inducer); and/or
nanovesicle drug carriers according to any one of claims 1-57; and/or nanoparticle drug carriers according to any one of claims 130-132; and/or
nanomaterial carriers according to any one of claims 133-137.
143. A formulation for inducing immunogenic cell death, said formulation comprising a nanomaterial that induces ICD.
144. The formulation of claim 143, wherein said nanomaterial contains or comprises a nanomaterial that induces ICD.
145. The formulation of claim 144, wherein said nanomaterial that induces ICD forms a nanoparticle.
146. The formulation according to any one of claims 143-145, wherein said nanomaterial comprises a material selected from the group consisting of CuO, Cu20, Sb203, As203, Bi203, P203, ZnO, Ti02, graphene oxide, and 2D materials other than graphene or graphene oxide.
PCT/US2018/033265 2017-05-18 2018-05-17 Nano-enabled immunotherapy in cancer WO2018213631A1 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU2018269742A AU2018269742B2 (en) 2017-05-18 2018-05-17 Nano-enabled immunotherapy in cancer
EP18803111.6A EP3624810A4 (en) 2017-05-18 2018-05-17 Nano-enabled immunotherapy in cancer
CA3063932A CA3063932A1 (en) 2017-05-18 2018-05-17 Nano-enabled immunotherapy in cancer
US16/687,368 US11433143B2 (en) 2017-05-18 2019-11-18 Nano-enabled immunotherapy in cancer
US17/823,044 US20230241244A1 (en) 2017-05-18 2022-08-29 Nano-enabled immunotherapy in cancer

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201762507996P 2017-05-18 2017-05-18
US62/507,996 2017-05-18
US201862614325P 2018-01-05 2018-01-05
US62/614,325 2018-01-05

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US16/687,368 Continuation-In-Part US11433143B2 (en) 2017-05-18 2019-11-18 Nano-enabled immunotherapy in cancer

Publications (1)

Publication Number Publication Date
WO2018213631A1 true WO2018213631A1 (en) 2018-11-22

Family

ID=64274728

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/033265 WO2018213631A1 (en) 2017-05-18 2018-05-17 Nano-enabled immunotherapy in cancer

Country Status (4)

Country Link
EP (1) EP3624810A4 (en)
AU (1) AU2018269742B2 (en)
CA (1) CA3063932A1 (en)
WO (1) WO2018213631A1 (en)

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110585131A (en) * 2019-09-20 2019-12-20 宁夏医科大学 Chemotherapy drug co-loaded 1-methyltryptophan immune prodrug micelle, preparation method and application thereof
WO2020072366A1 (en) * 2018-10-01 2020-04-09 The Regents Of The University Of California Methods for treating cancer with double stranded rna sensor activators and adoptive cell therapy
CN111012919A (en) * 2019-12-23 2020-04-17 山东大学 PEGylated ICD inducer-IDO inhibitor nanoconjugate, and preparation method and application thereof
CN111330004A (en) * 2020-03-04 2020-06-26 中国人民解放军军事科学院军事医学研究院 Application of molybdenum disulfide nanosheet layer in preparation of dendritic cell function promoter
CN111333653A (en) * 2019-12-16 2020-06-26 山东大学 ICD inducer-IDO inhibitor conjugate, preparation method and application
WO2021061837A1 (en) * 2019-09-23 2021-04-01 President And Fellows Of Harvard College Biomaterial-based antigen free vaccine and the use thereof
WO2021076630A1 (en) * 2019-10-14 2021-04-22 The Regents Of The University Of California Nano-enabled immunotherapy in cancer
WO2021096542A1 (en) 2019-11-12 2021-05-20 Nammi Therapeutics, Inc. Formulated and/or co-formulated liposome compositions containing ido antagonist prodrugs useful in the treatment of cancer and methods thereof
WO2021126281A1 (en) 2019-12-20 2021-06-24 Nammi Therapeutics, Inc. Formulated and/or co-formulated liposome compositions containing toll-like receptor ("tlr") agonist prodrugs useful in the treatment of cancer and methods thereof
WO2021167703A1 (en) 2020-02-19 2021-08-26 Nammi Therapeutics, Inc. Formulated and/or co-formulated liposome compositions containing tgfb antagonist prodrugs useful in the treatment of cancer and methods thereof
CN113332450A (en) * 2021-05-11 2021-09-03 铜仁学院 Preparation method of zinc oxide nanoparticles for eye drug delivery system, and use method and application thereof
CN113353939A (en) * 2021-05-25 2021-09-07 中国科学院上海硅酸盐研究所 Band gap adjustable and degradability controllable two-dimensional hydrosilylene nano material and preparation method and application thereof
WO2022010322A1 (en) * 2020-07-10 2022-01-13 (주)나디안바이오 Pharmaceutical composition for preventing or treating cancer comprising naphthoquinone-based compound and immune checkpoint inhibitor as active ingredients
WO2022031605A1 (en) * 2020-08-07 2022-02-10 The Regents Of The University Of California Combination chemo-immunotherapy for pancreatic cancer using the immunogenic effects of an irinotecan silicasome nanocarrier plus anti-pd-1
WO2022055542A1 (en) 2020-09-10 2022-03-17 Nammi Therapeutics, Inc. Formulated and/or co-formulated liposome compositions containing pd-1 antagonist prodrugs useful in the treatment of cancer and methods thereof
WO2022120344A1 (en) * 2020-12-01 2022-06-09 The Trustees Of Columbia University In The City Of New York Method of sensitizing cancers to immunotherapy using immunomodulatory agents
CN114748414A (en) * 2022-04-20 2022-07-15 中国医学科学院生物医学工程研究所 Sodium alginate hydrogel composite system for co-loading chemotherapeutic drug and nanoparticles and preparation method and application thereof
CN114890385A (en) * 2021-07-01 2022-08-12 中国科学院上海硅酸盐研究所 Efficient antioxidant two-dimensional hydrogermanium alkene nanosheet and preparation method and application thereof
US11433143B2 (en) 2017-05-18 2022-09-06 The Regents Of The University Of California Nano-enabled immunotherapy in cancer
WO2022197344A1 (en) 2021-03-18 2022-09-22 Nammi Therapeutics, Inc. Formulated and/or co-formulated liposome compositions containing a2ar antagonist prodrugs useful in the treatment of cancer and methods thereof
US11555177B2 (en) 2016-07-13 2023-01-17 President And Fellows Of Harvard College Antigen-presenting cell-mimetic scaffolds and methods for making and using the same
WO2023091168A1 (en) 2021-11-18 2023-05-25 Nammi Therapeutics, Inc. Formulated and/or co-formulated nanocarriers compositions containing immunogenic cell death (icd) inducing prodrugs useful in the treatment of cancer and methods thereof
CN116327979A (en) * 2023-05-25 2023-06-27 西南石油大学 Transition metal-based mesoporous nano catalytic medicine, preparation method and application
US11752238B2 (en) 2016-02-06 2023-09-12 President And Fellows Of Harvard College Recapitulating the hematopoietic niche to reconstitute immunity
WO2023172347A1 (en) 2022-03-10 2023-09-14 Nammi Therapeutics, Inc. Formulated and/or co-formulated lipid nanocarriers compositions containing toll-like receptor ("tlr") agonist prodrugs useful in the treatment of cancer and methods thereof
WO2023172300A1 (en) * 2022-03-10 2023-09-14 The Regents Of The University Of California A drug nanocarrier system to deliver a combination of tlr agonists and/or a lipoxin plus immunogenic cell death inducing chemotherapeutic agents for cancer immunotherapy
WO2023183872A1 (en) * 2022-03-23 2023-09-28 You First Services, Inc. Delmopinol and delmopinol salt containing nanoparticles and uses thereof
EP4083037A4 (en) * 2019-12-26 2024-01-24 Yonsei Univ Univ Industry Foundation Uif Pyrrolidine derivative, and pharmaceutical composition for preventing or treating beta-amyloid or tau protein-associated diseases containing same
EP4146274A4 (en) * 2020-05-04 2024-04-17 Drugcendr Australia Pty Ltd Methods for treating pancreatic cancer and other solid tumors

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014138278A1 (en) * 2013-03-05 2014-09-12 The Regents Of The University Of California Lipid bilayer coated mesoporous silica nanoparticles with a high loading capacity for one or more anticancer agents
WO2017201528A1 (en) * 2016-05-20 2017-11-23 The University Of Chicago Nanoparticles for chemotherapy, targeted therapy, photodynamic therapy, immunotherapy, and any combination thereof

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110159017A1 (en) * 2008-04-11 2011-06-30 Ludwig Institute For Cancer Research Ltd. Trytophan catabolism in cancer treatment and diagnosis
WO2017023667A1 (en) * 2015-07-31 2017-02-09 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Immunostimulatory nanocarrier
EP4309739A3 (en) * 2017-01-27 2024-04-17 The Methodist Hospital Core/shell structure platform for immunotherapy
WO2019173391A1 (en) * 2018-03-06 2019-09-12 Rita Elena Serda A high capacity platform for immunogenic cancer cell death

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014138278A1 (en) * 2013-03-05 2014-09-12 The Regents Of The University Of California Lipid bilayer coated mesoporous silica nanoparticles with a high loading capacity for one or more anticancer agents
WO2017201528A1 (en) * 2016-05-20 2017-11-23 The University Of Chicago Nanoparticles for chemotherapy, targeted therapy, photodynamic therapy, immunotherapy, and any combination thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
PURI ET AL.: "Lipid-Based Nanoparticles as Pharmaceutical Drug Carriers: From Concepts to Clinic", HHS PUBLIC ACCESS, 14 June 2010 (2010-06-14), pages 3, XP055580417, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2885142> *
See also references of EP3624810A4 *
SUN ET AL.: "Programmable co-delivery of the immune checkpoint inhibitor NLG919 and chemotherapeutic doxorubicin via a redox-responsive immunostimulatory polymeric prodrug carrier", ACTA PHARMACOLOGICA SINICA, vol. 38, 8 May 2017 (2017-05-08), pages 823 - 834, XP055580410 *
YAMANO ET AL.: "Whole cell vaccination using immunogenic cell death by an oncolytic adenovirus is effective against a colorectal cancer model", MOLECULAR THERAPY - ONCOLYTICS, vol. 4, 14 December 2016 (2016-12-14), pages 1 - 6, XP055580408 *

Cited By (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11752238B2 (en) 2016-02-06 2023-09-12 President And Fellows Of Harvard College Recapitulating the hematopoietic niche to reconstitute immunity
US11555177B2 (en) 2016-07-13 2023-01-17 President And Fellows Of Harvard College Antigen-presenting cell-mimetic scaffolds and methods for making and using the same
US11433143B2 (en) 2017-05-18 2022-09-06 The Regents Of The University Of California Nano-enabled immunotherapy in cancer
WO2020072366A1 (en) * 2018-10-01 2020-04-09 The Regents Of The University Of California Methods for treating cancer with double stranded rna sensor activators and adoptive cell therapy
CN110585131A (en) * 2019-09-20 2019-12-20 宁夏医科大学 Chemotherapy drug co-loaded 1-methyltryptophan immune prodrug micelle, preparation method and application thereof
WO2021061837A1 (en) * 2019-09-23 2021-04-01 President And Fellows Of Harvard College Biomaterial-based antigen free vaccine and the use thereof
WO2021076630A1 (en) * 2019-10-14 2021-04-22 The Regents Of The University Of California Nano-enabled immunotherapy in cancer
EP4045054A4 (en) * 2019-10-14 2023-11-29 The Regents of the University of California Nano-enabled immunotherapy in cancer
WO2021096542A1 (en) 2019-11-12 2021-05-20 Nammi Therapeutics, Inc. Formulated and/or co-formulated liposome compositions containing ido antagonist prodrugs useful in the treatment of cancer and methods thereof
CN115135385A (en) * 2019-11-12 2022-09-30 纳米医疗有限公司 Formulated and/or co-formulated liposomal compositions containing IDO antagonist prodrugs and methods thereof for treating cancer
CN111333653A (en) * 2019-12-16 2020-06-26 山东大学 ICD inducer-IDO inhibitor conjugate, preparation method and application
WO2021126281A1 (en) 2019-12-20 2021-06-24 Nammi Therapeutics, Inc. Formulated and/or co-formulated liposome compositions containing toll-like receptor ("tlr") agonist prodrugs useful in the treatment of cancer and methods thereof
US11744874B2 (en) 2019-12-20 2023-09-05 Nammi Therapeutics, Inc. Formulated and/or co-formulated liposome compositions containing toll-like receptor (“TLR”) agonist prodrugs useful in the treatment of cancer and methods thereof
WO2021129160A1 (en) * 2019-12-23 2021-07-01 山东大学 Nanoconjugate of pegylated icd inducer-ido inhibitor, preparation method therefor, and application thereof
CN111012919A (en) * 2019-12-23 2020-04-17 山东大学 PEGylated ICD inducer-IDO inhibitor nanoconjugate, and preparation method and application thereof
EP4083037A4 (en) * 2019-12-26 2024-01-24 Yonsei Univ Univ Industry Foundation Uif Pyrrolidine derivative, and pharmaceutical composition for preventing or treating beta-amyloid or tau protein-associated diseases containing same
WO2021167703A1 (en) 2020-02-19 2021-08-26 Nammi Therapeutics, Inc. Formulated and/or co-formulated liposome compositions containing tgfb antagonist prodrugs useful in the treatment of cancer and methods thereof
CN111330004B (en) * 2020-03-04 2023-02-10 中国人民解放军军事科学院军事医学研究院 Application of molybdenum disulfide nanosheet layer in preparation of dendritic cell function promoter
CN111330004A (en) * 2020-03-04 2020-06-26 中国人民解放军军事科学院军事医学研究院 Application of molybdenum disulfide nanosheet layer in preparation of dendritic cell function promoter
EP4146274A4 (en) * 2020-05-04 2024-04-17 Drugcendr Australia Pty Ltd Methods for treating pancreatic cancer and other solid tumors
WO2022010322A1 (en) * 2020-07-10 2022-01-13 (주)나디안바이오 Pharmaceutical composition for preventing or treating cancer comprising naphthoquinone-based compound and immune checkpoint inhibitor as active ingredients
WO2022031605A1 (en) * 2020-08-07 2022-02-10 The Regents Of The University Of California Combination chemo-immunotherapy for pancreatic cancer using the immunogenic effects of an irinotecan silicasome nanocarrier plus anti-pd-1
WO2022055542A1 (en) 2020-09-10 2022-03-17 Nammi Therapeutics, Inc. Formulated and/or co-formulated liposome compositions containing pd-1 antagonist prodrugs useful in the treatment of cancer and methods thereof
US11833209B2 (en) 2020-09-11 2023-12-05 Nammi Therapeutics, Inc. Formulated and/or co-formulated liposome compositions containing PD-1 antagonist prodrugs useful in the treatment of cancer and methods thereof
WO2022120344A1 (en) * 2020-12-01 2022-06-09 The Trustees Of Columbia University In The City Of New York Method of sensitizing cancers to immunotherapy using immunomodulatory agents
WO2022197344A1 (en) 2021-03-18 2022-09-22 Nammi Therapeutics, Inc. Formulated and/or co-formulated liposome compositions containing a2ar antagonist prodrugs useful in the treatment of cancer and methods thereof
CN113332450B (en) * 2021-05-11 2023-08-04 铜仁学院 Preparation method and application method of zinc oxide nanoparticles of ocular drug delivery system
CN113332450A (en) * 2021-05-11 2021-09-03 铜仁学院 Preparation method of zinc oxide nanoparticles for eye drug delivery system, and use method and application thereof
CN113353939B (en) * 2021-05-25 2023-09-08 中国科学院上海硅酸盐研究所 Band gap adjustable and degradability controllable two-dimensional hydrosilylene nano material and preparation method and application thereof
CN113353939A (en) * 2021-05-25 2021-09-07 中国科学院上海硅酸盐研究所 Band gap adjustable and degradability controllable two-dimensional hydrosilylene nano material and preparation method and application thereof
CN114890385B (en) * 2021-07-01 2023-09-08 中国科学院上海硅酸盐研究所 Efficient antioxidant two-dimensional hydrogen germanium alkene nano-sheet and preparation method and application thereof
CN114890385A (en) * 2021-07-01 2022-08-12 中国科学院上海硅酸盐研究所 Efficient antioxidant two-dimensional hydrogermanium alkene nanosheet and preparation method and application thereof
WO2023091168A1 (en) 2021-11-18 2023-05-25 Nammi Therapeutics, Inc. Formulated and/or co-formulated nanocarriers compositions containing immunogenic cell death (icd) inducing prodrugs useful in the treatment of cancer and methods thereof
WO2023172347A1 (en) 2022-03-10 2023-09-14 Nammi Therapeutics, Inc. Formulated and/or co-formulated lipid nanocarriers compositions containing toll-like receptor ("tlr") agonist prodrugs useful in the treatment of cancer and methods thereof
WO2023172300A1 (en) * 2022-03-10 2023-09-14 The Regents Of The University Of California A drug nanocarrier system to deliver a combination of tlr agonists and/or a lipoxin plus immunogenic cell death inducing chemotherapeutic agents for cancer immunotherapy
WO2023183872A1 (en) * 2022-03-23 2023-09-28 You First Services, Inc. Delmopinol and delmopinol salt containing nanoparticles and uses thereof
CN114748414A (en) * 2022-04-20 2022-07-15 中国医学科学院生物医学工程研究所 Sodium alginate hydrogel composite system for co-loading chemotherapeutic drug and nanoparticles and preparation method and application thereof
CN116327979A (en) * 2023-05-25 2023-06-27 西南石油大学 Transition metal-based mesoporous nano catalytic medicine, preparation method and application

Also Published As

Publication number Publication date
CA3063932A1 (en) 2018-11-12
AU2018269742A1 (en) 2019-12-05
EP3624810A1 (en) 2020-03-25
EP3624810A4 (en) 2021-02-17
AU2018269742B2 (en) 2024-01-11

Similar Documents

Publication Publication Date Title
AU2018269742B2 (en) Nano-enabled immunotherapy in cancer
US20230241244A1 (en) Nano-enabled immunotherapy in cancer
Wang et al. Tumor penetrability and anti-angiogenesis using iRGD-mediated delivery of doxorubicin-polymer conjugates
Arabi et al. Targeting CD44 expressing cancer cells with anti-CD44 monoclonal antibody improves cellular uptake and antitumor efficacy of liposomal doxorubicin
US10335420B2 (en) Nanoparticle-based tumor-targeted drug delivery
Guo et al. Liposomal nanoparticles carrying anti-IL6R antibody to the tumour microenvironment inhibit metastasis in two molecular subtypes of breast cancer mouse models
Waelti et al. Targeting her-2/neu with antirat Neu virosomes for cancer therapy
AU2020367786A1 (en) Nano-enabled immunotherapy in cancer
US20230338363A1 (en) Combination chemo-immunotherapy for pancreatic cancer using the immunogenic effects of an irinotecan silicasome nanocarrier plus anti-pd-1
Huang et al. PEGylated immunoliposome-loaded endoglin single-chain antibody enhances anti-tumor capacity of porcine α1, 3GT gene
TW201804987A (en) Ephrin receptor A2 (EphA2)-targeted docetaxel-generating nano-liposome compositions
US20220378936A1 (en) Delivery system complexes comprising a precipitate of an active agent and methods of use
EP2228059A1 (en) Chemotherapeutic composition for the treatment of cancer
US20210100791A1 (en) Combined pharmaceutical formulation comprising drug-containing liposome composition and immune checkpoint inhibitor
JP5817053B2 (en) Liposomes with tumor specificity
Gupta et al. Immunoliposomes: a targeted drug delivery system for cancer therapeutics and vaccination
US20230398077A1 (en) Silicasome nanocarrier for metal-based drug delivery
Kim et al. Oral Cancer Immunotherapy through a Simvastatin‐Loaded Colloidal Dispersion System for the Generation of Sustained Antitumor Immunity
WO2023172300A1 (en) A drug nanocarrier system to deliver a combination of tlr agonists and/or a lipoxin plus immunogenic cell death inducing chemotherapeutic agents for cancer immunotherapy
WO2023081514A1 (en) Cxcr4 antagonist loaded liposomes and silicasomes
Gu et al. Dual‐Targeting Nanoliposome Improves Proinflammatory Immunomodulation of the Tumor Microenvironment
Brown Anti-GD2 Etoposide-Loaded Immunoliposomes for the Treatment of GD2 Positive Tumors

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18803111

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3063932

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2018269742

Country of ref document: AU

Date of ref document: 20180517

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2018803111

Country of ref document: EP

Effective date: 20191218