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WO2025076398A1 - Conjugaison immunomodulatrice de nanoparticules et ses utilisations - Google Patents

Conjugaison immunomodulatrice de nanoparticules et ses utilisations Download PDF

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Publication number
WO2025076398A1
WO2025076398A1 PCT/US2024/050023 US2024050023W WO2025076398A1 WO 2025076398 A1 WO2025076398 A1 WO 2025076398A1 US 2024050023 W US2024050023 W US 2024050023W WO 2025076398 A1 WO2025076398 A1 WO 2025076398A1
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nanoparticle
immunomodulatory
cells
antigen
ligands
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Inventor
Michelle S. BRADBURY
Ulrich Wiesner
Nabil SIDDIQUI
Gabriel DeLeon
Jedd D. Wolchok
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Cornell University
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Cornell University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4256—Tumor associated carbohydrates
    • A61K40/4257—Mucins, e.g. MUC-1
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    • 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
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1241—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins
    • A61K51/1244—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • This invention relates generally to combinatorial methods and compositions for the treatment of a disease or condition [e.g., cancer, e.g., ovarian cancer, e.g., relapsed and refractory ovarian cancer, e.g., breast cancer, e.g., colon cancer, e.g., lung cancer, e.g., pancreas cancer, endometrial cancer, e.g., benign conditions such as ovarian cysts, endometriosis, pancreatitis, inflammatory disease (e.g., pelvic inflammatory disease), early pregnancy, hepatitis, cirrhosis, or pericarditis] in subjects. More specifically, in certain embodiments, the invention relates to enhanced particle-driven therapies for combinatorial treatment of a disease or condition by targeting mucin- 16 (MUC16) in combination with engineered cellular therapy (e.g., CAR T cell therapy).
  • MUC16 mucin- 16
  • engineered cellular therapy
  • the invention is directed to a method of treating a disease or condition [e.g., cancer, e.g., ovarian cancer, e.g., relapsed and refractory ovarian cancer, e.g., breast cancer, e.g., colon cancer, e.g., lung cancer, e.g., pancreas cancer, endometrial cancer, e.g., benign conditions such as ovarian cysts, endometriosis, pancreatitis, inflammatory disease (e.g., pelvic inflammatory disease), early pregnancy, hepatitis, cirrhosis, or pericarditis], the method comprising: administering to a subject (1) a first pharmaceutical composition comprising (a) an immunomodulatory nanoparticle conjugate, wherein the immunomodulatory nanoparticle conjugate comprises: (i) a nanoparticle; and (ii) a plurality of cellular receptor- and/or antigen- targeting ligands (e.
  • a disease or condition
  • the plurality of cellular receptor- and/or antigen- targeting ligands comprises one or more species of targeting ligands.
  • the plurality of ODNs bind to Toll-like receptor 9 (TLR9) (e.g., thereby promoting expression of co-stimulatory molecules, secretion of inflammatory cytokines, and/or development of CD8+ T cell responses).
  • TLR9 Toll-like receptor 9
  • the plurality of cellular receptor- and/or antigen- targeting ligands comprises an anti-MUC16 antibody fragment (e.g., anti-MUC16 scFv) (e.g., conjugated to the nanoparticle).
  • an anti-MUC16 antibody fragment e.g., anti-MUC16 scFv
  • conjugated to the nanoparticle e.g., conjugated to the nanoparticle.
  • the antibody fragment or scFv comprises a first linker sequence
  • the first linker sequence comprises a sequence having at least 80% identity to, e.g., at least 85% identity to, e.g., at least 90% identity to, e.g., at least 95% identity to, e.g., at least 98% identity to, e.g., at least 100% identity to GGGGSGGGGSGGGGS (SEQ ID NO: 3).
  • the scFv comprises a second linker sequence attached (e.g., covalently or non-covalently) to the C-terminus end of the scFv.
  • the second linker sequence comprises a cysteine residue.
  • the second linker sequence comprises a sequence having at least 85% identity to, e.g., at least 100% identity to GGSCGSG (SEQ ID NO: 3).
  • the plurality of cellular receptor- and/or antigen- targeting ligands comprises from 1 to 75 cellular receptor- and/or antigen- targeting ligands (e.g., of a single species) (e.g., 1 to 50 ligands, e.g., 2 to 50, e.g., 5 to 30 ligands, e.g., about 6 to 8 ligands, e.g., about 1 to 5 ligands, e.g., about 1 to 2 ligands) conjugated to the nanoparticle.
  • 1 to 50 ligands e.g., 2 to 50, e.g., 5 to 30 ligands, e.g., about 6 to 8 ligands, e.g., about 1 to 5 ligands, e.g., about 1 to 2 ligands
  • the immunomodulatory nanoparticle conjugate comprises a radiotherapeutic agent (e.g., a radiolabel, e.g., a radioisotope, e.g., 225-Actinium) (e.g., formulated at a therapeutically effective dose, e.g., at least 0.27 microcuries/kg, e.g., at least 1.0 microcuries/kg, e.g., at least 2 microcuries/kg, e.g., at least 2.7 microcuries/kg, e.g., relative to a microenvironment control).
  • a radiotherapeutic agent e.g., a radiolabel, e.g., a radioisotope, e.g., 225-Actinium
  • a therapeutically effective dose e.g., at least 0.27 microcuries/kg, e.g., at least 1.0 microcuries/kg, e.g., at least 2 microcuries/kg, e.g
  • the immunomodulatory nanoparticle conjugate has an average diameter no greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm).
  • the nanoparticle comprises a silica composition such that ferroptosis is not induced (e.g., ferroptosis is switched “off’).
  • the nanoparticles have a silica composition such that ferroptosis may be induced (e.g., ferroptosis is not switched “off’).
  • the immunomodulatory nanoparticle conjugate further comprises a therapeutic agent (e.g., wherein the therapeutic agent is attached to the nanoparticle, or to the plurality of cellular receptor- and/or antigen- targeting ligands, or to both the nanoparticle and the plurality of cellular receptor- and/or antigen- targeting ligands, e.g., wherein the attachment is covalent or non-covalent).
  • a therapeutic agent e.g., wherein the therapeutic agent is attached to the nanoparticle, or to the plurality of cellular receptor- and/or antigen- targeting ligands, or to both the nanoparticle and the plurality of cellular receptor- and/or antigen- targeting ligands, e.g., wherein the attachment is covalent or non-covalent).
  • the nanoparticle further comprises a targeting ligand.
  • the first pharmaceutical composition and/or second pharmaceutical composition further comprises a carrier.
  • the engineered cells are MUC 16-targeting engineered cells.
  • the first pharmaceutical composition is being used to enhance activity of the second pharmaceutical composition.
  • the first pharmaceutical composition activates anti-tumor immune responses in the tumor microenvironment (TME), e.g., without off-target effects.
  • TEE tumor microenvironment
  • the first pharmaceutical composition is administered before, after, or at the same time as the second pharmaceutical composition.
  • the invention is directed to a immunomodulatory nanoparticle conjugate comprising: a nanoparticle; and a plurality of cellular receptor- and/or antigen- targeting ligands (e.g., anti-MUC16 antibody fragment, oligodeoxynucleotides (ODNs), e.g., CpG-ODNs) conjugated to the nanoparticle, wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., as measurement by fluorescence correlation spectroscopy, e.g., saline solution) (e.g., wherein the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g.
  • DLS dynamic light scatter
  • the invention is directed a composition (e.g., pharmaceutical composition, e.g., a radiotherapeutic composition) comprising an immunomodulatory nanoparticle conjugate and engineered cells (e.g., engineered immune cells, e.g., CAR T cells, e.g., wherein the engineered cells are or comprise MUC16-targeting engineered cells, e.g., wherein the engineered cells secrete IL-12, e.g., wherein the engineered cells are or comprise MUC16-targeting, IL-12 secreting CAR T cells) for use in engineered cellular therapy (e.g., CAR T cell therapy) (e.g., wherein the immunomodulatory nanoparticle conjugate is being used to enhance activity of the engineered cells) (e.g., wherein the immunomodulatory nanoparticle conjugate activates anti-tumor immune responses in the tumor microenvironment (TME), e.g., without off-target effects).
  • engineered cells e.g., engineered immune cells,
  • the plurality of ODNs comprises or is a plurality of ODNs containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)), and wherein the CpG motif comprises or is a sequence according to 5’-TCCATGACGTTCCTGACGTT-3’ (SEQ ID NO: 4).
  • CpG cytosine-phosphate-guanine
  • the immunomodulatory nanoparticle conjugate further comprises a therapeutic agent (e.g., wherein the therapeutic agent is attached to the nanoparticle, or to the plurality of immunomodulatory/stimulatory ligands, or to both the nanoparticle and the plurality of immunomodulatory/stimulatory ligands, e.g., wherein the attachment is covalent or non-covalent).
  • a therapeutic agent e.g., wherein the therapeutic agent is attached to the nanoparticle, or to the plurality of immunomodulatory/stimulatory ligands, or to both the nanoparticle and the plurality of immunomodulatory/stimulatory ligands, e.g., wherein the attachment is covalent or non-covalent.
  • the plurality of immunomodulatory/stimulatory ligands conjugated to the nanoparticle enhance immunomodulatory properties of the nanoparticle (e.g., properties present in the nanoparticle even in the absence of the immunomodulatory ligands).
  • the plurality of immunomodulatory/stimulatory ligands does or does not modulate the tumor microenvironment.
  • the method comprises administering a therapeutic radioisotope (e.g., wherein the therapeutic radioisotope is attached to a second nanoparticle having a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., as measurement by fluorescence correlation spectroscopy, e.g., saline solution) (e.g., wherein the radioisotope is attached to the nanoparticle or the second nanoparticle via a second chelator)) (e.g., wherein the second nanoparticle has a diameter from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm).
  • a therapeutic radioisotope
  • FIG. 1 is an illustrative schematic of MUC16 showing a retained MUC16 ecto domain and a 4H11 mAb binding site of a retained portion of MUC16 (arrow).
  • FIG. 5A shows a HPLC graph of anti-MUC16 scFv-PEG-C’ dots (upper panel) and PEG-Cy5-C’ dots (lower panel).
  • FIG. 6A shows C’ dot modulation of the transcriptome based on genotypic status through the use of bulk RNA-seq analyses of murine ID8(L), MP, and MPB1 cells exposed to C’ dots (15pM, 24h), normalized to saline vehicle.
  • FIG. 7B shows cytokine/chemokine profiles derived from culture media of particle- vs saline-exposed cells (from FIG. 7A) treated after 24h.
  • FIG. 8A shows viability of luc+ ID8(L), ID8(H), MP, MPB1 cells 24h after incubation with (i) media alone (“Control”), (ii) C’ dots alone (15 pM, 24h; “C’ dots”) alone or (iii) followed by co-culture with anti-MUC16-CAR T cells (1 : 1 E:T) cell ratio) for 24h; “C’ dots + CAR T cells”), or (iv) anti-MUC16-CAR T cells alone (E:T 1 : 1; “CAR T Cells”).
  • FIG. 8C shows viability of luc+ ID8(L) 24h after incubation following the same treatments as in FIG. 8A, except using MUC16-targeting C’ dots.
  • FIG. 9E shows fluorescence imaging of ex vivo organs harvested from control mice (y- axis on color scale shows radiant efficiency).
  • FIG. 10D shows cytokine profiles of ascites specimens.
  • FIG. 10E shows persistence of CAR T cells in blood.
  • FIG. HA shows single cell dissociates of ID8(L) tumor specimens stained for surface markers at 4 or 10 days after CAR T cell or vehicle administration for CD8+ T cells.
  • FIG. 11B shows single cell dissociates of ID8(L) tumor specimens stained for surface markers at 4 or 10 days after CAR T cell or vehicle administration for CAR T cells.
  • FIG. 11C shows single cell dissociates of ID8(L) tumor specimens stained for surface markers at 4 or 10 days after CAR T cell or vehicle administration for Tregs.
  • FIG. 13B shows a volcano plot of differentially expressed genes between C’ dot and vehicle-treated samples in tumor cells.
  • FIG. 13C shows a volcano plot of differentially expressed genes between C’ dot and vehicle-treated samples in macrophages.
  • FIG. 13F shows gene set enrichment analysis of genes significantly up- (red-blue) or down-regulated (green) in tumor cells; y-axis shows the enrichment score, x-axis shows rank in ordered data set for enrichment profile (green), hits (black), or ranking metric scores (light grey).
  • FIG. 13G shows gene set enrichment analysis of genes significantly up- (red-blue) or down-regulated (green) in tumor cells; y-axis shows the enrichment score, x-axis shows rank in ordered data set for enrichment profile (green), hits (black), or ranking metric scores (light grey).
  • FIG. 13H shows gene set enrichment analysis of genes significantly up- (red-blue) or down-regulated (green) in tumor cells; y-axis shows the enrichment score, x-axis shows rank in ordered data set for enrichment profile (green), hits (black), or ranking metric scores (light grey).
  • FIG. 15B shows characterization of CpG-PEG-Cy5-C' dots using a gel permeation chromatography (GPC) elugram.
  • FIG. 16A shows BMDCs cultured with media alone, lOOnM PEG-C’ dots, or lOOnM CpG-C’ dots for 48 h and analyzed by flow cytometry for % dendritic cells (%DCs). *p ⁇ 0.05, ***p ⁇ 0.005, ****p ⁇ 0.001; by one-way ANOVA.
  • FIG. 16E shows RT-qPCR of BMDCs treated as in FIG. 15 A.
  • FIG. 16G shows BMDCs exposed to CpG-C’ dots (red) and lOkD dextran (green) showing colocalization (yellow) by super-resolution microscopy. Scale bai- 10pm.
  • FIG. 16H shows a phagocytosis assay of NP-exposed BMDCs co-cultured with B 16- GM cells for 48h. *p ⁇ 0.05, ***p ⁇ 0.005, ****p ⁇ 0.001; by one-way ANOVA.
  • CpG-C’ dots (6 nmoles (lOpM), red), as against PEG-C’ dots (gray, dotted line), and saline vehicle (blue), initiated when tumors reached ⁇ 50 mm 3 (day 0), and repeated on days 3 and 6 thereafter.
  • FIG. 18C shows the ratio of M1/M2 macrophages in due to CpG-C’ dot treatment in B 16-GM tumor mice triply injected with CpG-C’ dots or C’ dots (6 nmoles; lOpM) or saline vehicle (control) every 3 days when tumor volumes reached 50mm 3 .
  • FIG. 18D shows the population of Treg cells as % CD45+ cells due to CpG-C’ dot treatment in B16-GM tumor mice triply injected with CpG-C’ dots or C’ dots (6 nmoles; lOpM) or saline vehicle (control) every 3 days when tumor volumes reached 50mm 3 .
  • Antibody refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature are approximately 150 kD tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure.
  • Each heavy chain is comprised of at least four domains (each about 110 amino acids long)- an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHI, CH2, and the carboxy-terminal CH3 (located at the base of the Y’s stem).
  • VH amino-terminal variable
  • CH2 amino-terminal variable
  • CH3 carboxy-terminal CH3
  • Each light chain is comprised of two domains - an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another “switch”.
  • Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed.
  • Naturally-produced antibodies are also glycosylated, typically on the CH2 domain.
  • the Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity.
  • affinity and/or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification.
  • antibodies produced and/or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation.
  • antibody agent refers to an agent that specifically binds to a particular antigen.
  • the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding.
  • Exemplary antibody agents include, but are not limited to monoclonal antibodies or polyclonal antibodies.
  • an antibody agent may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies.
  • an antibody agent may include one or more sequence elements are humanized, primatized, chimeric, etc., as is known in the art.
  • Antigen' refers to an agent that elicits an immune response; and/or (ii) an agent that binds to a T cell receptor e.g., when presented by an MHC molecule) or to an antibody.
  • an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies); in some embodiments, an elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen).
  • an antigen binds to an antibody and may or may not induce a particular physiological response in an organism.
  • Biocompatible refers to materials that do not cause significant harm to living tissue when placed in contact with such tissue, e.g., in vivo. In certain embodiments, materials are “biocompatible” if they are not toxic to cells. In certain embodiments, materials are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death. In certain embodiments, materials are biodegradable.
  • Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocar
  • Wilms tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.
  • HCC hepatocellular cancer
  • lung cancer e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non small cell lung cancer (NSCLC), adenocarcinoma of the lung
  • Identity refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided sequences are known in the art.
  • Calculation of the percent identity of two nucleic acid or polypeptide (e.g., amino acid) sequences can be performed by aligning the two sequences (or the complement of one or both sequences) for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position.
  • a marker is a cytokine.
  • a marker is a chemokine.
  • a marker is a receptor.
  • a marker is a genetic marker (e.g., mRNA, RNA) indicative of activation of a gene.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspension
  • Radiolabel refers to a moiety comprising a radioactive isotope of at least one element. Exemplary suitable radiolabels include but are not limited to those described herein. In certain embodiments, a radiolabel is one used in positron emission tomography (PET). In certain embodiments, a radiolabel is one used in single-photon emission computed tomography (SPECT).
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • subject includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses).
  • subjects are mammals, particularly primates, especially humans.
  • subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats.
  • subject mammals will be, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like.
  • treatment refers to administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
  • such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
  • such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
  • treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.
  • compositions, and processes are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited steps.
  • the immunomodulatory nanoparticle conjugates comprise an ultrasmall (e.g., sub-50 nm diameter, e.g., sub-20 nm diameter, e.g., sub- 15 nm diameter, e.g., sub- 10 nm diameter, e.g., sub-8 nm diameter) silica nanoparticle containing a deep red/near-infrared dye (e.g., Cy5; absorption peak: 650 nm) that is covalently encapsulated within the silica-matrix.
  • a deep red/near-infrared dye e.g., Cy5; absorption peak: 650 nm
  • the brightness is dramatically improved (e.g., at least 2-times, e.g., at least 10- times, e.g., at least 50-times, e.g., at least 100-times, e.g., at least 600-times) as compared to the free dye.
  • cellular binding ligands can be attached to the nanoparticle as described herein.
  • the immunomodulatory nanoparticle conjugates comprise from 1 to 100 discrete cellular receptor- and/or antigen- targeting ligands (e.g., of the same type or different types), wherein the cellular receptor- and/or antigen- targeting ligands bind to receptors on tumor cells (e.g., wherein the immunomodulatory nanoparticle conjugates have an average diameter no greater than 15 nm, e.g., no greater than 10 nm, e.g., from about 5 nm to about 7 nm, e.g., about 6 nm).
  • radiolabels and/or fluorescent markers attached to (or incorporated in or on, or otherwise associated with) the nanoparticles provide quantitative assessment of immunomodulatory nanoparticle conjugates uptake at the target site and within the body, as well as permit monitoring of treatment response.
  • modular linkers are described for incorporating targeting ligands to develop a drug delivery system with controlled pharmacological properties.
  • the described platforms determine the influence of targeting on immunomodulatory nanoparticle conjugates penetration and accumulation, thereby establishing an adaptable platform for improved delivery of a range of tractable SMIs, for example, to primary and metastatic brain tumors.
  • PET Pierismography
  • tracers are used as imaging agents.
  • fluorophores comprise Dil, DiR, DiD, and the like.
  • Fluorochromes comprise far red, and near infrared fluorochromes (NIRF).
  • Fluorochromes include but are not limited to a carbocyanine and indocyanine fluorochromes.
  • imaging agents comprise commercially available fluorochromes including, but not limited to methylene blue, Cy5.5, Cy5 and Cy7 (GE Healthcare); AlexaFlour660, AlexaFlour680, AlexaFluor750, and AlexaFluor790 (Invitrogen); VivoTag680, VivoTag-S680, and VivoTag-S750 (VisEn Medical); Dy677, Dy682, Dy752 and Dy780 (Dyomics); DyLight547, DyLight647 (Pierce); HiLyte Fluor 647, HiLyte Fluor 680, and HiLyte Fluor 750 (AnaSpec); IRDye 800CW, IRDye 800RS, and IRDye 700DX (Li-Cor); methylene blue; and ADS780WS, ADS830WS, and ADS832WS (American Dye Source) and Kodak X-SIGHT 650, Kodak X-SIGHT 691, Ko
  • a multi-wavelength camera as described by Bradbury et al. US Publication No. US 2015/0182118 Al, “Systems, Methods, and Apparatus for Multichannel Imaging of Fluorescent Sources in Real Time”, the disclosure of which is hereby incorporated by reference in its entirety.
  • the imaging system used to image the lesion provides both static and functional assessments of the area of treatment (and its surroundings).
  • the nanoconjugate comprises more than one chelator.
  • immunomodulatory nanoparticle conjugates are comprised of intrinsically therapeutic ultrasmall silica nanoparticles to which a plurality of immunomodulatory/stimulatory ligands (e.g., a plurality of pattern recognition receptors (PRR) agonist moieties (e.g., a plurality of oligodeoxy nucleotides (ODNs)) containing an unmethylated cytosine- phosphate-guanine (CpG) motif (or CpG ODNs))) can be conjugated thereto.
  • a plurality of immunomodulatory/stimulatory ligands e.g., a plurality of pattern recognition receptors (PRR) agonist moieties (e.g., a plurality of oligodeoxy nucleotides (ODNs)) containing an unmethylated cytosine- phosphate-guanine (CpG) motif (or CpG ODNs)
  • the described base nanoparticles themselves exhibit significant intrinsic proinflammatory activities (e.g., activities that modulate macrophage phenotype, e.g., via augmentation of interferons that stimulate the immune system) within the tumor microenvironment.
  • proinflammatory activities e.g., activities that modulate macrophage phenotype, e.g., via augmentation of interferons that stimulate the immune system
  • CpG immune checkpoint blockade
  • systemic administration of the described nanoparticle with or without immunomodulatory/stimulatory ligands attached thereto in certain models can functionally serve as a potent exogenous agonist of multiple pattern recognition receptors or PRRs, such as STING-, TLR-, and IFN-related pathways, and offer the potential to activate tumor cells, as well as innate and adaptive immune cell populations, within the tumor microenvironment (TME).
  • PRRs multiple pattern recognition receptors or PRRs
  • PRRs such as STING-, TLR-, and IFN-related pathways
  • the described base nanoparticle itself shows enhanced immunologic responses (e.g., affecting upregulation of the R1G/MDA5-MAVS axis) in ovarian models treated systemically.
  • Such effects can be enhanced by attachment of a plurality of immunomodulatory/stimulatory ligands to the base nanoparticle.
  • Other variables that can influence immunologic responses described herein include size of the nanoparticle, material composition of the nanoparticles, and surface chemistry of the nanoparticle, as described herein. Nanomedicine strategies for targeting suppressive myeloid cells and limiting resistance.
  • TEE tumor microenvironment
  • NPs biocompatible nanoparticles
  • TLR Toll-like receptor
  • PEGylated, Cy5-dye encapsulating and aMSH-ligand bearing targeted fluorescent coreshell silica nanoparticles e.g., aMSH-PEG-Cy5-C’ dots
  • aMSH-PEG-Cy5-C’ dots e.g., aMSH-PEG-Cy5-C’ dots
  • PEG-Cy5-C’ dots e.g., PEG-Cy5-C’ dots
  • Cy5-maleimido derivatives is first coupled to a mercapto-silane to form a dye-silane conjugate.
  • the dye-silane conjugate is subsequently co-condensed with TMOS in aqueous solutions at basic pH to form the Cy5 dye-encapsulating silica core.
  • silica particle growth is quenched at appropriate time intervals to control silica core size by adding either monofunctional PEG-silane (6-9 EO units per chain), resulting in untargeted PEG-Cy5-C’ dots.
  • first hetero-bifunctional PEG, functionalized on one end with a silane and on the other with aMSH peptide, immediately followed by monofunctional PEG-silane is used to quench the reaction.
  • the nanoparticle comprises aMSH ligands.
  • ligand density is varied between 5 and 15 ligands per particle by adding increasing amounts of heterobifunctional PEG to the growing silica cores.
  • subsequent purification from unreacted precursors and/or particle aggregates is performed using gel permeation chromatography (GPC).
  • fluorescence correlation spectroscopy determines particle hydrodynamic size and concentration.
  • Dynamic light scattering (DLS) and/or zeta-potential measurements determine hydrodynamic size and/or surface charge.
  • UV-VIS absorption and emission spectroscopy determine a number of dyes and/or aMSH ligands per particle (e.g., in conjunction with FCS).
  • Transmission electron microscopy (TEM) determines silica core size. Controlling Hydrophobic Particle Surface Patchiness
  • control over the surface patchiness can be exerted by controlling the number of Cy5 dyes on the surface of the silica core of a nanoparticle by using different concentrations of ammonia as sol-gel catalyst. In certain embodiments, there are between zero and four Cy5 dyes on the silica core surface. In certain embodiments, patchiness has an effect on ferroptosis induction. In certain embodiments, patchiness has an effect on immune cell priming and/or activation. Hydrophobic patchiness from Cy5 dyes ending up on the C’ dot surface can be verified by HPLC. For example, a HPLC using 150 mm Waters Xbridge BEH C4 protein separation columns with 300 A pore size and 3.5 pm particle size, and a water/acetonitrile mixture as mobile phase may be used.
  • Cy5 dye-silane conjugate can be co-condensed with tetramethoxy- silane (TMOS) and two other precursors (e.g., aluminum-tri-sec-butoxide (Al(OBu s )3) / 3-(trihydroxysilyl)propyl methylphosphonate (TPMP) for Al / P incorporation) at appropriate pH to form a Cy5-encapsulating core.
  • TMOS tetramethoxy- silane
  • Al(OBu s )3 aluminum-tri-sec-butoxide
  • TPMP trihydroxysilylpropyl methylphosphonate
  • affinity of iron to a silica core is modulated through phosphonate- silane conjugates co-condensed with TMOS in the silica core synthesis.
  • Phosphonates are known for their high affinity to metal ions like iron. Beyond about 15 mole% of phosphonate-silane in the reaction, relative to TMOS, the effect of ferroptosis on amino-acid-deprived MDA-MB-468 TNBCs at C’ dot concentrations of 15 is essentially switched off. Without wishing to be bound to any particular theory, this is due to the high affinity of iron to the phosphonate groups and related reduction of iron release once the iron-loaded particles are internalized by cells.
  • phosphonate group bearing C’ dots effect ferroptosis and/or immune cell priming and/or activation.
  • microwave plasma atomic emission spectroscopy is used to evaluate nanoparticle iron concentrations. These nanoparticles help delineate molecular mechanisms by which C’ dots induce ferroptosis and/or activation of immune cells.
  • compositional and/or structural parameters of silica nanoparticles e.g., PEG-Cy5-C’ dots or C’ dots
  • TEE tumor microenvironment
  • Example 2 Synthesis, characterization, and purification of MUC16-targeting C’ dots.
  • a linker has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, a linker has a length of 6 amino acids. In some embodiments, a linker has a length of 7 amino acids. In some embodiments, a linker has a length of 10 amino acids. In some embodiments, a linker has a length of 15 amino acids. In some embodiments, a linker has a length of 20 amino acids.
  • FIG. 3 shows concentration and time-dependent gene expression modulation by C’ dots in ID8 cells.
  • a heatmap of gene expression profiles of ID8 cells incubated with increasing C’ dot concentrations (0, 0.1, 1, 15 pM) is shown over a 6-day period. Each quadrant represents an independent biological replicate.
  • MUC16 ecto + ID8 cell lines can be conducted using MUC16 ecto + ID8 cell lines, CAR T cells, bone marrow derived macrophages (BMDMs) and dendritic cells exposed to selected non-targeting and MUC16- targeting particles (as described herein) in order to identify lead candidates that enhance anti-tumor immune cell cytotoxicity and ferroptosis.
  • BMDMs bone marrow derived macrophages
  • dendritic cells exposed to selected non-targeting and MUC16- targeting particles (as described herein) in order to identify lead candidates that enhance anti-tumor immune cell cytotoxicity and ferroptosis.
  • MUC16+ MP cells substantially higher uptake (factor 10-fold at lOOnM) was noted for anti-hMUC16 scFv-PEG-C’ dots relative to plain C’ dots, unlike that found for non-MUC16-expressing (MUC16-) MP cells.
  • cellular internalization was greater for the targeted particle probe over a 24-h period by confocal microscopy (FIGs. 5C and 5D). No significant loss of viability was observed (data not shown), as demonstrated in our prior uptake studies using different cancer cell types.
  • MUC16-targeting C’ dots also significantly enhanced representative proinflammatory cytokine/chemokine secretion over non-targeting C’ dots (FIG. 5E).
  • Reactome in FIG. 6B revealed significant genotype- specific regulation of pro-inflammatory, immunogenic, and cytotoxic signatures.
  • Relative to ID8(L) cells, MP and/or MPB1 cells revealed upregulation of TN Fa, cytokine/chemokine signaling, protein transcription and translation, and cell cycle checkpoint pathways. Induction of metabolic and ferroptotic (cytotoxic) pathways was significantly more pronounced in ID8(L) cells.
  • genes representing five major functional classes were selected for expression profiling in cell lines.
  • C’ dots may induce a cell stress response in OC cells, for example, through ferroptotic induction, as evidenced by a significant increase in Hsp70 mRNA in vitro and in vivo (see FIG. 7A; FIG. 10B).
  • Sustained transcriptional activation of cytokines correlated with increased cytokine secretion profiles (FIG. 7B), which were significantly upregulated in MP and MPB1 cells.
  • FIG. 7B Sustained transcriptional activation of cytokines correlated with increased cytokine secretion profiles
  • FIG. 7B cytokine secretion profiles
  • CAR T cells IL- 12 secreting CAR T cells
  • any engineered immune cells e.g., engineered T cells, beyond the exemplified MUC16- targeting, IL- 12 secreting CAR T cells may be used in combination with the provided immunomodulatory nanoparticle conjugates
  • a series of co-culture experiments were performed with CAR T cells and luciferase (/wc+j-expressing OC cells, the latter with or without particle pre- treatment.
  • FIG. 8C shows competitive binding and/or viability of luc+ ID8(L) 24h after incubation following the same treatments as in (a), except using MUC16-targeting C’ dots.
  • Imaging techniques described herein can also be conducted using the immunomodulatory nanoparticle conjugates (e.g., MUC16-targeting nanoparticles) described herein.
  • FIG. 9G demonstrate a high tumor-to-background ratio on maximum intensity projection (M1P) PET imaging of ID8 tumor-bearing mice (3 wks post-inoculation) 24 hours p.i. of 89 Zr-DFO-PEG-C dots.
  • Particle dose escalation procedures (up to 6 doses) can be implemented, as needed, at levels that induce the foregoing responses (e.g., 12 nmoles/dose, 60 M stock): (i) maximum fold changes (i.e., >2) for specific genes (e.g., cytokines and DAMPs), (ii) increased cytotoxic T cell activity (j'CD8+/Treg), (iii) reduced immune suppression (JM1/M2, J, percentage (%) of Tregs, MDSCs in tumors), and (iv) enhanced cytotoxicity will serve as the initial dosing level for MP and MPB 1 models with the aim of maximizing efficacy, as described herein.
  • maximum fold changes i.e., >2
  • specific genes e.g., cytokines and DAMPs
  • JM1/M2 reduced immune suppression
  • JM1/M2 percentage (%) of Tregs, MDSCs in tumors
  • enhanced cytotoxicity will serve as the initial dosing level for
  • Immunophenotyping can be performed on tumor, ascites, and splenic tissue specimens harvested from ID8 models for both particle- and vehicle-treated cohorts.
  • a multiparametric How cytometry (MPFC) panel can be used to analyze and compare populational changes in immune cell infiltrates.
  • MPFC multiparametric How cytometry
  • the following endpoints can be used: (i) innate immune responses, (ii) cytotoxic T cell activity ()CD8+/Trcg), and (iii) reduced immune suppression ( M1/M2, J, Tregs, MDSC) within tumors.
  • the CD8+/Treg ratio increased 100-fold, largely due to a near complete loss of Tregs over this time period. This was accompanied by significant and sustained increases in co-stimulatory molecules (i.e., CD40 and CD86) on Ml macrophages and the M 1/M2 ratio, as well as an acute increase in the overall DC population. Factor of 3 decreases in MDSCs were also seen over this 10-day period (FIG. 12A-12F).
  • particle concentrations can be increased from 60 pM to lOOpM and dosing frequencies can be re-evaluated (i.e., 1, 3, 6 doses).
  • Preliminary studies e.g., FIG. 5 show that particle concentration is one of the major drivers of ferroptosis and immunomodulation. Accordingly, increasing particle concentration can further augment these effects to potentially improve treatment of immune suppressive TMEs.
  • FIGs. i 1 A- HE Confirmation of a role for particle-mediated Treg decreases (see FIGs. i 1 A- HE) can be investigated by conducting IP and Treg transfer studies in depleted regulatory T cell (DEREG) mice. This can allow for a distinction between Treg loss of phenotypic/functional lineage commitment and selective depletion of Treg cells.
  • DEREG depleted regulatory T cell
  • BMDCs Bone marrow-derived dendritic cells
  • BMDCs bone marrow-derived dendritic cells
  • FIG. 16A % MHC-II-positive (+) DCs
  • FIG. 16B MHC-II protein expression
  • FIG. 16D Tlr9+ DCs
  • FIG. 16E Marked increases in specific DC maturation/co- stimulatory pathway markers
  • FIG. 16F Marked increases in anti-tumor cytokine production

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Abstract

Sont divulgués dans la présente invention des conjugués de nanoparticules immunomodulatrices utiles pour des agents thérapeutiques et/ou des diagnostics. Dans certains modes de réalisation, chaque conjugué de nanoparticules immunomodulatrices comprend une nanoparticule qui présente une pluralité de ligands de ciblage de récepteur cellulaire et/ou d'antigène (par exemple, la pluralité de ligands de ciblage de récepteur cellulaire et/ou d'antigène ciblant des domaines qui sont exprimés sur la surface de cellules tumorales, par exemple, la pluralité de ligands de ciblage de récepteur cellulaire et/ou d'antigène ciblant le domaine MUC16ecto exprimé sur la surface de cellules tumorales, par exemple, la pluralité de ligands de ciblage de récepteur cellulaire et/ou d'antigène contenant un anti-MUC16, par exemple, la pluralité de ligands de ciblage de récepteur cellulaire et/ou d'antigène comprenant une pluralité de ligands immunomodulateurs/stimulateurs (de la même espèce ou d'espèces différentes) (par exemple, une pluralité d'oligodésoxynucléotides (ODNs) (par exemple, un ODN contenant un motif cytosine-phosphate-guanine (CpG) non méthylé (ou des ODN CpG)) conjugué à la nanoparticule) conjugué à la nanoparticule.
PCT/US2024/050023 2023-10-05 2024-10-04 Conjugaison immunomodulatrice de nanoparticules et ses utilisations Pending WO2025076398A1 (fr)

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Publication number Priority date Publication date Assignee Title
US20150076392A1 (en) * 2012-01-23 2015-03-19 Nvigen, Inc. Low Density, Highly Porous Nano Structure
US20200297854A1 (en) * 2012-06-07 2020-09-24 President And Fellows Of Harvard College Nanotherapeutics for drug targeting
US20210269500A1 (en) * 2015-08-12 2021-09-02 Massachusetts Institute Of Technology Cell surface coupling of nanoparticles
US10317395B1 (en) * 2015-08-31 2019-06-11 Cornell University Ex vivo engineered immune organoids for controlled germinal center reactions
WO2022271619A1 (fr) * 2021-06-21 2022-12-29 Memorial Sloan-Kettering Cancer Center Amélioration médiée par des nanoparticules d'une immunothérapie pour favoriser la cytotoxicité induite par ferroptose et les réponses immunitaires antitumorales

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