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WO2025038450A1 - Immunothérapies à particules à base de cellules immunitaires ultra-petites, compositions de cellules immunitaires et leurs utilisations - Google Patents

Immunothérapies à particules à base de cellules immunitaires ultra-petites, compositions de cellules immunitaires et leurs utilisations Download PDF

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Publication number
WO2025038450A1
WO2025038450A1 PCT/US2024/041718 US2024041718W WO2025038450A1 WO 2025038450 A1 WO2025038450 A1 WO 2025038450A1 US 2024041718 W US2024041718 W US 2024041718W WO 2025038450 A1 WO2025038450 A1 WO 2025038450A1
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nanoparticle
immunomodulatory
immune cell
cell engager
targeting ligands
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WO2025038450A9 (fr
Inventor
Michelle S. BRADBURY
Nabil SIDDIQUI
Gabriel DeLeon
Ulrich Wiesner
Feng Chen
Jedd D. Wolchok
Taha MERGHOUB
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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • This invention relates generally to methods and compositions for the treatment of diseases in subjects. More specifically, in certain embodiments, the invention relates to engineered immune cells (e.g., combined with silica nanoparticles) for localized targeted therapy (e.g., combinatorial therapy) for treatment of diseases such as cancer, brain injury (e.g., radiation, trauma), neurological disorders, inflammatory diseases, or other indications.
  • engineered immune cells e.g., combined with silica nanoparticles
  • targeted therapy e.g., combinatorial therapy
  • diseases such as cancer, brain injury (e.g., radiation, trauma), neurological disorders, inflammatory diseases, or other indications.
  • the described technology employs engineered immunomodulatory particle compositions and immunotherapies for localized targeted therapy and improved properties (such as increased half life in the bloodstream) in the treatment of cancer and other diseases, injuries, or conditions.
  • the described immunomodulatory particle compositions comprise ultrasmall silica nanoparticle compositions associated with a plurality of immune cell engager targeting ligands conjugated to the nanoparticle.
  • the plurality of immune cell engager targeting ligands may be linked (e.g., covalently, e.g., non-covalently) to each other as described herein (e.g., BiTE targeting ligands).
  • the immune cell engager targeting ligands may not be linked to each other as described herein (e.g., dual targeting ligands).
  • the described immunomodulatory particle compositions can be used alone or in combination with engineered immunotherapies comprised of engineered immune cells.
  • the described engineered immune cells comprise chimeric antigen receptors (CARs) and secrete bispecific immune cell engagers (e.g., bispecific T cell Engagers, “BiTEs”, i.e., “CAR.BiTE engineered immune cells”).
  • CARs chimeric antigen receptors
  • BiTEs bispecific T cell Engagers
  • the CAR.bispecific immune cell engager (e.g., CAR.BiTE) engineered immune cell targets a diseased cell antigen or receptor, and then the CAR releases the bispecific immune cell engager (e.g., the BiTE).
  • the bispecific immune cell engager activates immune cells through its cognate receptor while the disease-targeting moiety (e.g., scFv) binds its respective cognate receptor, in turn, promoting enhanced cytotoxic responses relative to each one alone.
  • the bispecific immune cell engager engages immune cells or heterogeneous tumor cells, depending on the design of the BiTE, at the site of the diseased cell.
  • the CAR.bispecific immune cell engager e.g., CAR.BiTE engineered immune cells are combined with immunomodulatory silica nanoparticle conjugates (e.g., with or without the immune cell engager (targeting ligands e.g., BiTE targeting ligands, e.g., dual targeting ligands) attached to the particles) for combinatorial treatment of a disease or condition.
  • the silica nanoparticles may also have other targeting ligands and/or therapeutics attached thereto for modulating the microenvironment.
  • Immune-modulating silica particles described herein can be engineered to create a variety of topologies, including spherical, ring, or cage conformations.
  • Immune cells beyond T cells can be engineered to express CARs for a particular therapy.
  • the CAR-immune cell can be a CAR-T cell, a CAR-macrophage, a CAR-neutrophil, or CAR-NK cells, or other cell type.
  • CAR.bispecific immune cell engagers e.g., CAR.BiTE
  • CAR T.BiTE CAR T.BiTE
  • different CAR.bispecific immune cell engagers e.g., CAR.BiTE
  • CAR T.BiTE CAR T.BiTE
  • different types of targeted immunomodulatory silica nanoparticles e.g., BiTE-functionalized silica nanoparticles, e.g., CpG-functionalized silica nanoparticles (as an adjuvant)
  • targeted silica topologies e.g., C ring, e.g., cage
  • the described engineered immunotherapies comprise CpG oligonucleotides covalently coupled with single-chain fragments (e.g., FcyRII/CD32 on antigen presenting cells or “APCs”) silica nanoparticles as an adjuvant (see, e.g., Sepulveda- Toepfer et al., Human Vaccines & Immunotherapies, 2019, Vol. 15, No. 1, 179-188, the contents of which is hereby incorporated by reference in its entirety).
  • the described engineered immunotherapies comprise CpG alone (e.g., not conjugated to a nanoparticle) as an adjuvant.
  • Nanoparticles with varying composition e.g., aluminum-containing, e.g., aC’ dots
  • methods of using the described engineered immunotherapies can improve treatment outcomes under conditions of diseased-induced immunosuppression and immune resistance by harnessing the favorable biological properties of a set of self-therapeutic ultrasmall (e.g., diameter less than 8 nm) silica organic core-shell nanoparticles with tunable size, composition, surface chemical properties, and functionalities.
  • the bispecific immune cell engager (e.g., BiTE) component addresses several limiting factors, including those related to heterogeneous expression of disease antigens (e.g., tumor antigens), lack of immune cell infiltration (e.g., cold tumor microenvironment), and/or suppressive T cell exhaustion markers.
  • the described engineered immunotherapies can also be adapted to include a synthetic NOTCH or other tumor recognition “on/off ’ switch (e.g., chimeric antigen receptor, MESA, GEMs) that can improve issues related to specificity and heterogeneity to increase therapeutic benefits of CAR T cells.
  • a synthetic NOTCH or other tumor recognition “on/off ’ switch e.g., chimeric antigen receptor, MESA, GEMs
  • the described engineered immunotherapies can also be applied to diseases beyond cancer, including brain injury (e.g., radiation, trauma) and neurological disorders by targeted immune cell engagers (e.g., BiTEs) to be secreted by the CAR-immune cells.
  • brain injury e.g., radiation, trauma
  • targeted immune cell engagers e.g., BiTEs
  • the invention is directed to an immunomodulatory nanoparticle conjugate comprising: a nanoparticle; and a plurality of multispecific immune cell engager targeting ligands (e.g., bispecific T-cell engagers or “BiTEs”, e.g., bispecific macrophage engagers, e.g., bispecific neutrophil engagers, e.g., dual (discrete) targeting ligands, e.g., trispecific killer engagers or “TriKEs”, e.g., bifunctional checkpoint-inhibitory T cell engagers or “CiTEs”, e.g., simultaneous multiple interaction T cell engagers or “SMITEs”) conjugated to the nanoparticle (e.g., wherein the multispecific immune cell engager targeting ligands are associated with each other; e.g., wherein the multispecific immune cell engager targeting ligands are not associated with each other, e.g., wherein the multispecific immune cell engager targeting ligands
  • the average nanoparticle diameter is from 1 to 20 nm (e g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm, e.g., from 4 to 8 nm).
  • the immunomodulatory nanoparticle conjugate has an average diameter no greater than 50 nm, no greater than 40 nm, no greater than 30 nm, no greater than 20 nm, no greater than 15 nm, no greater than 10 nm.
  • the nanoparticle comprises silica.
  • the nanoparticle comprises aluminum (e.g., an aC’ dot).
  • the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in an aqueous solution (e.g., a saline solution).
  • DLS dynamic light scattering
  • the plurality of multispecific immune cell engager targeting ligands are of at least two species (e.g., same or different species).
  • one or more of the plurality of multispecific immune cell engager targeting ligands are nanobodies.
  • the plurality of multispecific immune cell engager targeting ligands comprise: (1) a first cellular binding moiety that targets an immune cell-specific moiety; and (2) a second cellular binding moiety that targets antigens expressed by tumor cells and/or proteins expressed in inflammatory states, including neurodegenerative disorders or brain injury (e.g., radiation, trauma) (e.g., wherein the first and the second cellular binding moieties are not linked together).
  • bispecific T-cell engagers or “BiTEs” e.g., bispecific macrophage engagers, e.g., bispecific neutrophil engagers, e.g., dual (discrete) targeting ligands, e.g., trispecific killer engagers or “TriKEs”, e.g., bifunctional checkpoint-inhibitory T cell engagers or “CiTEs”, e.g., simultaneous multiple interaction T cell engagers or “SMITEs”
  • SMITEs simultaneous multiple interaction T cell engagers or “SMITEs”
  • the second cellular binding moiety targets PSMA and/or TYPR1.
  • the first cellular binding moiety comprises a toll-like receptor (TLR) agonist moiety (e g., an oligodeoxynucleotides (ODN), e.g., an ODN containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)).
  • TLR9 Toll-like receptor 9
  • first and the second cellular binding moieties are linked together.
  • the first and the second cellular binding moieties are not linked together.
  • the plurality of multispecific immune cell engager targeting ligands comprises a plurality of immune-related proteins (e.g., of at least two different species).
  • the plurality of multispecific immune cell engager targeting ligands comprises a plurality of antibody fragments (e.g., of at least two species) (e.g., linked together, not linked together).
  • the plurality of multispecific immune cell engager targeting ligands comprises a plurality of oligonucleotides (e.g., of at least two species) (e.g., linked together, not linked together).
  • the plurality of antibody fragments comprise a single chain variable fragment (scFv) (e.g., TYPR1 scFv).
  • scFv single chain variable fragment
  • the plurality of antibody fragments comprise a nanobody.
  • the plurality of multispecific immune cell engager targeting ligands comprise a CD3 targeting moiety, a CD8 targeting moiety, TYRP1 scFv, or a combination thereof.
  • the plurality of multispecific immune cell engager targeting ligands is or comprises a bispecific macrophage engager (BiME) targeting ligand and wherein the macrophage is activated by a signal regulatory protein-a (SIRP a) inhibitory antibody (e.g., wherein the antibody is directed to a particular tumor via a tumor associated antigen (TAA) antibody).
  • SIRP a signal regulatory protein-a
  • TAA tumor associated antigen
  • the plurality of multispecific immune cell engager targeting ligands is covalently or non-covalently bonded to the nanoparticle via a linker (e.g., a flexible linker (e.g., a glycine-serine linker), a cleavable linker (e.g., an enzymatically cleavable linker)) or covalently (e.g., via click chemistry, or non-covalently bonded directly to the nanoparticle) or associated with the nanoparticle or a moiety surrounding the nanoparticle (e.g., via van der Waals forces).
  • a linker e.g., a flexible linker (e.g., a glycine-serine linker), a cleavable linker (e.g., an enzymatically cleavable linker)
  • covalently e.g., via click chemistry, or non-covalently bonded directly to the nanoparticle
  • the nanoparticle is coated with an organic polymer (e.g., a partial or complete coating).
  • an organic polymer e.g., a partial or complete coating
  • the organic polymer is polyethylene glycol (PEG).
  • the immunomodulatory nanoparticle conjugate comprises a chelator.
  • the plurality of multispecific immune cell engager targeting ligands comprises from 1 to 75 multispecific immune cell engager targeting ligands conjugated to the nanoparticle.
  • the multispecific immune cell engager targeting ligands are of a single species.
  • the plurality of multispecific immune cell engager targeting ligands comprises from 1 to 50 multispecific immune cell engager targeting ligands conjugated to the nanoparticle. [0040] In certain embodiments, the plurality of multispecific immune cell engager targeting ligands comprises from 2 to 50 immune cell engager targeting ligands conjugated to the nanoparticle.
  • the plurality of multispecific immune cell engager targeting ligands comprises from 5 to 30 multispecific immune cell engager targeting ligands conjugated to the nanoparticle.
  • the plurality of multispecific immune cell engager targeting ligands comprises from about 6 to about 8 multispecific immune cell engager targeting ligands conjugated to the nanoparticle.
  • the plurality of multispecific immune cell engager targeting ligands comprises from about 1 to about 5 multispecific immune cell engager targeting ligands conjugated to the nanoparticle.
  • the plurality of multispecific immune cell engager targeting ligands comprises from about 1 to 2 multispecific immune cell engager targeting ligands conjugated to the nanoparticle.
  • the immunomodulatory nanoparticle conjugate comprises (e.g., is additionally modified by) a radiolabel.
  • the nanoparticle comprises silica.
  • the nanoparticle comprises a silica core.
  • the nanoparticle comprises a silica core and a silica shell surrounding at least a portion of the core.
  • the nanoparticle comprises a fluorescent compound within the core.
  • the immunomodulatory nanoparticle conjugate further comprises a therapeutic agent.
  • the therapeutic agent is (i) attached to the nanoparticle, or (ii) to the plurality of immunomodulatory/ stimulatory ligands, or (iii) to both the nanoparticle and the plurality of immunomodulatory/stimulatory ligands (e.g., wherein the attachment is covalent or non- covalent).
  • the immunomodulatory nanoparticle conjugate further comprises a targeting ligand attached to the nanoparticle.
  • the immunomodulatory nanoparticle further comprises an immunomodulatory/stimulatory ligand attached to the nanoparticle (e.g., not as part of the multispecific immune cell engager targeting ligand) (e.g., not as part of the multispecific immune cell engager targeting ligand) [e.g., a toll-like receptor (TLR) agonist moiety, e.g., a pattern recognition receptor (PRR) agonist moiety, e.g., an oligodeoxynucleotides (ODN), e.g., an ODN containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)] [wherein the immunomodulatory/stimulatory ligand conjugated to the nanoparticle enhances immunomodulatory properties of the nanoparticle (e.g., properties present in the nanoparticle even in the absence of the immunomodulatory/stimulatory ligand)].
  • TLR toll-like receptor
  • PRR pattern recognition
  • the plurality of multispecific immune cell engager targeting ligands conjugated to the nanoparticle enhance immunomodulatory properties of the nanoparticle (e.g., properties present in the nanoparticle even in the absence of the multispecific immune cell engager targeting ligands).
  • the plurality of multispecific immune cell engager targeting ligands switches on or off (e.g., substantially upregulates or downregulates, e.g., by a factor of at least 50 at least 100 or at least 150) one or more specific cell populations in the tumor microenvironment (e.g., wherein the plurality of multispecific immune cell engager targeting ligands switches on or off regulatory T cell (Treg) population in the tumor microenvironment).
  • Treg regulatory T cell
  • the multispecific immune cell engager targeting ligands are immunostimulatory (e.g., the multispecific immune cell engager targeting ligands enhance ongoing immune responses).
  • the invention is directed to an engineered immunotherapy [e.g., for localized treatment, e.g., for combinatorial therapy, e.g., for treatment of cancer (e.g., solid primary tumors, e.g., metastatic tumors) (e.g., melanoma, e.g., rare melanoma, e.g., acral melanoma), brain injury (e.g., radiation, trauma), neurological disorders, inflammatory diseases, autoimmune diseases, or other diseases] comprising: (1) an engineered immune cell [e.g., a macrophage, e.g., a neutrophil, e.g., a T cell (e.g., a Steapl CAR-T cell), e.g., an NK cell] comprising (a) a chimeric antigen receptor (CAR) and (b) a multispecific immune cell engager (e.g., bispecific T-cell engagers
  • CAR chimeric
  • the bispecific multispecific immune cell engager (e.g., bispecific T-cell engager or “BiTE”, e.g., bispecific macrophage engager, e.g., bispecific neutrophil engager, e.g., dual (discrete) targeting ligand, e.g., trispecific killer engager or “TriKE”, e.g., bifunctional checkpoint-inhibitory T cell engager or “CiTE”, e g., simultaneous multiple interaction T cell engager or “SMITE”) comprises a plurality of cellular binding moi eties [e.g., an immunomodulatory/ stimulatory ligand, e.g., a single-chain variable fragments (scFv), e.g., nanobodies].
  • scFv single-chain variable fragments
  • the plurality of cellular binding moieties comprises: a first cellular binding moiety that binds to an immune cell-specific target (e.g., CD3, e.g., CD8, e.g., a pattern recognition receptor (PRR)); and a second cellular binding moiety that binds to a disease- associated antigen [e.g., a tumor-associated antigen (e.g., PSMA, e.g., TYPR1), e.g., an inflammatory- associated antigen (e.g., an antigen associated with diabetes), e.g., a neurological disease-associated antigen, e.g., a brain injury-associated antigen] [e.g., wherein the first cellular binding moiety and the second cellular binding moiety are attached by a linker (e.g., a flexible linker)].
  • a linker e.g., a flexible linker
  • the first cellular binding moiety is or comprises a toll-like receptor (TLR) agonist moiety (e.g., an oligodeoxynucleotides (ODN), e.g., an ODN containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)).
  • TLR toll-like receptor
  • the plurality of CpG 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 engineered immune cell further comprises a synthetic “on/off” system (e.g., a synthetic NOTCH system).
  • a synthetic “on/off” system e.g., a synthetic NOTCH system
  • the engineered immunotherapy further comprises oligodeoxynucleotide (ODN) (e.g., an ODN containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)) (e.g., not conjugated to the nanoparticle, e.g., conjugated to the nanoparticle).
  • ODN oligodeoxynucleotide
  • CpG cytosine-phosphate-guanine
  • CpG ODNs CpG ODNs
  • the invention is directed to an engineered immunotherapy [e.g., for localized therapy, e.g., for combinatorial therapy, e.g., for treatment of cancer (e.g., solid tumors, e.g., metastatic tumors, e.g., melanoma, e.g., rare melanoma, e.g., acral melanoma), brain injury (e.g., radiation, trauma), neurological disorders, inflammatory diseases, or other diseases] comprising: (1) an engineered immune cell [e.g., a macrophage, e.g., a neutrophil, e.g., a T cell (e.g., a Steapl CAR-T cell), e.g., an NK cell] comprising a chimeric antigen receptor (CAR), and a multispecific immune cell engager (e.g., BiTE) [e.g., wherein the multispecific immune cell engager (CAR), and a multi
  • the invention is directed to a method (e.g., a combinatorial method) of treating a disease or condition, the method comprising administering (e.g., systemically) to a subject a pharmaceutical composition comprising the immunomodulatory nanoparticle conjugate of any embodiment described herein, or the engineered immune cell of any embodiment described herein, or the engineered immunotherapy of any embodiment described herein (e.g., to target a particular type of tissue, e.g., cancer (e.g., melanoma, e.g., rare melanoma, e.g., acral melanoma, e.g., brain cancer, e.g., breast cancer, e.g., ovarian cancer, e.g., tumor metastases), e.g., brain injury (e.g., radiation, trauma), e.g., inflammatory disease, e.g., neurological disorder, e.g., autoimmune disease).
  • a pharmaceutical composition comprising the
  • the invention is directed to a combinatorial method of treating a disease or condition, the method comprising administering(e.g., systemically) (e.g., simultaneously, e.g., separately) to a subject: (1) the immunomodulatory nanoparticle conjugate of any embodiment described herein (e.g., to target a particular type of tissue, e.g., cancer (e.g., melanoma, e.g., rare melanoma, e.g., acral melanoma, e.g., brain cancer, e.g., breast cancer, e.g., ovarian cancer, e.g., tumor metastases), e.g., brain injury (e.g., radiation, trauma), e.g., inflammatory disease, e.g., neurological disorder, e.g., autoimmune disease); and (2) an engineered immunotherapy of any embodiment described herein.
  • cancer e.g., melanoma, e.g
  • the invention is directed to a combinatorial method of treating a disease or condition, the method comprising administering (e.g., systemically) (e.g., simultaneously, e.g., separately) to a subject: (1) an engineered immunotherapy (e.g., as set forth herein) comprising a first engineered immune cell; and (2) a nanoparticle (e.g., wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) (e.g., wherein the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to
  • administering e.
  • the method further comprises administering a second engineered immune cell (e.g., wherein the second engineered immune cell is different than the first engineered immune cell, e.g., different type of immune cell, e.g., different type of CAR, e.g., different type of multispecific immune cell engager (e.g., bispecific T-cell engager or “BiTE”, e.g., bispecific macrophage engager, e.g., bispecific neutrophil engager, e.g., dual (discrete) targeting ligand, e.g., trispecific killer engager or “TriKE”, e.g., bifunctional checkpoint-inhibitory T cell engager or “CiTE”, e.g., simultaneous multiple interaction T cell engager or “SMITE”)) (e.g., at the same time, e.g., sequentially).
  • a second engineered immune cell e.g., wherein the second engineered immune cell is different than the first engineered immune cell, e.g
  • the method further comprises administering a toll receptor agonist moiety (e.g., an oligodeoxynucleotide (ODN) (e.g., an ODN containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)) (e.g., not conjugated to a nanoparticle, e.g., conjugated to a nanoparticle).
  • a toll receptor agonist moiety e.g., an oligodeoxynucleotide (ODN) (e.g., an ODN containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)
  • ODN oligodeoxynucleotide
  • CpG unmethylated cytosine-phosphate-guanine
  • CpG ODNs CpG ODNs
  • the immunomodulatory nanoparticle conjugate, and/or the engineered immune cell, and/or the engineered immunotherapy modulates the tumor microenvironment.
  • the immunomodulatory nanoparticle conjugate, and/or the engineered immune cell, and/or the engineered immunotherapy does not/do not modulate the tumor microenvironment.
  • the multispecific immune cell engager and/or the plurality of multispecific immune cell engager targeting ligands does or does not modulate the tumor microenvironment.
  • the multispecific immune cell engager e g., BiTE
  • the plurality of multispecific immune cell engager targeting ligands e.g., bispecific T-cell engagers or “BiTEs”, e.g., bispecific macrophage engagers, e.g., bispecific neutrophil engagers, e.g., dual (discrete) targeting ligands, e.g., trispecific killer engagers or “TriKEs”, e.g., bifunctional checkpoint-inhibitory T cell engagers or “CiTEs”, e.g., simultaneous multiple interaction T cell engagers or “SMITEs”), the immunomodulatory/ stimulatory targeting ligand, or any combination thereof, activates one or more pattern recognition receptors (PRRs) (e.g., toll-like receptors) (e.g., thereby activating different pro- inflammatory transcription factors (e.g., nuclear factor kappa B or NF-KB)
  • PRRs pattern recognition receptors
  • the method comprises administering a therapeutic radioisotope (e.g., wherein the therapeutic radioisotope is attached to a second nanoparticle having a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) (e.g., wherein the radioisotope is attached to the 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 e.g., wherein the therapeutic radioisotope is attached to
  • the pharmaceutical composition further comprises a carrier.
  • the method comprises administering one or more doses of the immunomodulatory nanoparticle conjugate or the nanoparticle (e.g., at different time points) (e.g., wherein the one or more doses of the immunomodulatory nanoparticle conjugate or the nanoparticle are administered prior to administering the engineered immunotherapy).
  • the administration of the one or more doses of the immunomodulatory nanoparticle conjugate or the nanoparticle modulates (e.g., “switches” on or off, e.g., substantially upregulates or downregulates, e.g., by a factor of at least 50 at least 100 or at least 150) one or more specific cell populations in the tumor microenvironment (e.g., “switches” on or off regulatory T cell (Treg) population in the tumor microenvironment).
  • the invention is directed to a method of in vivo imaging (e.g., image- guided intraoperative imaging) and/or therapy, the method comprising: administering to a subject a composition comprising the immunomodulatory nanoparticle conjugate of any embodiment described herein, or the engineered immune cell of any embodiment described herein, or the engineered immunotherapy of any embodiment described herein (e.g., such that the engineered immunotherapy preferentially collects in a particular region, e.g., near or within a particular tissue type, e.g., cancer, e.g., melanoma, e.g., rare melanoma, e.g., metastatic lymph node(s), e.g., brain injury (e g., radiation, trauma), e.g., neurological or pathological condition, e.g., neuroinflammatory disease, e.g., autoimmune disease), wherein the engineered immunotherapy is comprised of an imaging contrast label (e.g., associated
  • the invention is directed to a method of making the immunomodulatory nanoparticle conjugate of any embodiment described herein, the method comprising: contacting an aluminum containing compound with a protein-maleimide, thereby producing the immunomodulatory nanoparticle conjugate.
  • the method further comprises reacting an azide moiety (e.g., azide-PEG3-malemide linker) with a cysteine on the C-terminal region of the protein, thereby producing an azide-containing protein; and contacting the azide-containing protein with DBCO- functionalized nanoparticle, thereby producing a nanoparticle conjugate.
  • an azide moiety e.g., azide-PEG3-malemide linker
  • the method further comprises reacting an azide moiety (e.g., azide-PEG5-DOTA linker) with 177 Lu.
  • an azide moiety e.g., azide-PEG5-DOTA linker
  • the invention is directed to a method of making the immunomodulatory nanoparticle conjugate of any embodiment described herein, the method comprising: modifying the multispecific immune cell engager targeting ligand (protein) (e.g., BiTE targeting ligand) with a first click reactive group (e.g., an azide-moiety); modifying a nanopaiticle-PEG with a click partner of the first click reactive group (e.g., DBCO); and reacting the modified multi specific immune cell engager targeting ligand (protein) with the modified nanoparticle-PEG, thereby producing the immunomodulatory nanoparticle conjugate.
  • a first click reactive group e.g., an azide-moiety
  • a nanopaiticle-PEG e.g., DBCO
  • reacting the modified multi specific immune cell engager targeting ligand (protein) with the modified nanoparticle-PEG thereby producing the immunomodulatory nanoparticle conjugate.
  • the invention is directed to a composition
  • an engineered immune cell comprising an engineered immune cell (e.g., a macrophage, e.g., a neutrophil, e.g., a T cell, e.g., an NK cell) comprising a chimeric antigen receptor (CAR); and a multispecific immune cell engager (e.g., bispecific T-cell engager, “BiTE”, e.g., a bispecific macrophage engager, e.g., a bispecific neutrophil engager); and (2) a nanoparticle optionally having a plurality of multispecific immune cell engager targeting ligands (e.g., bispecific T-cell engagers or “BiTEs”, e.g., bispecific macrophage engagers, e.g., bispecific neutrophil engagers, e.g., dual (discrete) targeting ligands, e.g., trispecific killer engagers
  • an engineered immune cell comprising
  • the invention is directed to a composition
  • an engineered immune cell comprising an engineered immune cell (e.g., a macrophage, e.g., a neutrophil, e.g., a T cell, e.g., an NK cell) comprising a chimeric antigen receptor (CAR); and a multispecific immune cell engager (e.g., bispecific T-cell engager or “BiTE”, e.g., bispecific macrophage engager, e.g., bispecific neutrophil engager, e.g., dual (discrete) targeting ligand, e.g., trispecific killer engager or “TriKE”, e.g., bifunctional checkpoint-inhibitory T cell engager or “CiTE”, e.g., simultaneous multiple interaction T cell engager or “SMITE”); and (2) a nanoparticle optionally having a plurality of multispecific immune cell engager targeting ligands (e.g., bispecific
  • the invention is directed to a composition
  • an engineered immune cell comprising an engineered immune cell (e.g., a macrophage, e.g., a neutrophil, e.g., a T cell, e.g., an NK cell) comprising a chimeric antigen receptor (CAR); and a multispecific immune cell engager (e.g., bispecific T-cell engager or “BiTE”, e.g., bispecific macrophage engager, e.g., bispecific neutrophil engager, e.g., dual (discrete) targeting ligand, e.g., trispecific killer engager or “TriKE”, e.g., bifunctional checkpoint-inhibitory T cell engager or “CiTE”, e.g., simultaneous multiple interaction T cell engager or “SMITE”); and (2) a nanoparticle optionally having a plurality of multispecific immune cell engager targeting ligands (e.g., bispecific
  • FIG. 1 is an illustrative schema of screening steps for identifying candidate therapies.
  • FIG. 2 is an illustrative schema for testing immunotherapies described herein on a humanized AM (acryl melanoma) model.
  • FIG. 3 is an illustrative schema for testing immunotherapies as described herein.
  • FIG. 4 shows staining results for single cell dissociates of ID8(L) tumor specimens stained for surface markers at 4 or 10 days after CAR T cell or vehicle administration.
  • FIG. 4 Panel a shows results for CD8+ T cells.
  • FIG. 4 Panel b shows results for CAR T cells.
  • FIG. 4 Panel c shows results for Tregs.
  • FIG. 4 Panel d shows results for CXCR3+CAR T cells.
  • administration typically refers to the administration of a composition comprising a nanoparticle to a subject or system.
  • any route of administration may be utilized including, for example, parenteral (e.g., intravenous), oral, topical, systemic, subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into body compartments.
  • administration is oral. Additionally or alternatively, in certain embodiments, administration is parenteral. In certain embodiments, administration is intravenous. In certain embodiments, administration is intraperitoneal.
  • agent may refer to a compound, molecule, or entity of any chemical and/or biological class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof.
  • a small molecule polypeptide
  • nucleic acid for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof.
  • agent may refer to a compound, molecule, or entity that comprises a polymer. In certain embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In certain embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety. In some embodiments, the term may refer to a nanoparticle.
  • Antibody refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen.
  • intact antibodies as produced in nature are approximately 150 kD tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure.
  • Each heavy chain is comprised of at least four domains (each about 110 amino acids long)- an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHI, CH2, and the carboxy -terminal CH3 (located at the base of the Y’s stem).
  • VH amino-terminal variable
  • CHI amino-terminal variable
  • CH2 amino-terminal variable
  • CH3 located at the base of the Y’s stem
  • a short region known as the “switch” connects the heavy chain variable and constant regions.
  • the “hinge” connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody.
  • Each light chain is comprised of two domains - an amino-terminal variable (VL) domain, followed by a carboxy -terminal constant (CL) domain, separated from one another by another “switch”.
  • Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed.
  • Naturally-produced antibodies are also glycosylated, typically on the CH2 domain.
  • Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel.
  • Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4).
  • CDR1, CDR2, and CDR3 three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4).
  • 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.
  • any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and/or used as an “antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology.
  • an antibody is polyclonal; in some embodiments, an antibody is monoclonal.
  • an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies.
  • antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art.
  • an antibody utilized in accordance with certain embodiments of the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM ); single chain or Tandem diabodies (SIPsTM ); single chain or Tandem diabodies (SIPsTM ); single chain or Tandem diabodies (SIPsTM ); single chain or Tandem diabodies (SIPsTM ); single chain or Tandem
  • 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.
  • an antibody agent utilized in accordance with certain embodiments of the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM ); single chain or Tandem diabodies (TandAb®); VHH
  • an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally.
  • an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload or other pendant group).
  • an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody.
  • CDR complementarity determining region
  • an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR.
  • an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR.
  • an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR.
  • an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR.
  • an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain.
  • an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an Immunoglobulin-binding domain.
  • 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.
  • Antigen presenting cell The phrase “antigen presenting cell” or “APC ,” as used herein, has its art understood meaning referring to cells which process and present antigens to T-cells. Exemplary antigen cells include dendritic cells, macrophages and certain activated epithelial cells.
  • 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.
  • cancer refers to a malignant neoplasm or tumor (Stedman’s Medical Dictionary, 2 5t h ed.; Hensly ed.; Williams & Wilkins: Philadelphia, 1990).
  • 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
  • myelofibrosis MF
  • chronic idiopathic myelofibrosis chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEPNET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Page
  • Cellular binding ligand In general, the term “cellular binding ligand” is used herein to refer to any entity that binds to a target of interest as described herein (e.g., a target that is expressed on the surface of cells, e.g., expressed on the surface of T cells). In many embodiments, a cellular binding ligand of interest is one that binds specifically with its target in that it discriminates its target from other potential binding partners in a particular interaction context. In general, a cellular binding ligand may be or comprise an entity of any chemical class (e.g., polymer, non-polymer, small molecule, polypeptide, carbohydrate, lipid, nucleic acid, etc.).
  • a chemical class e.g., polymer, non-polymer, small molecule, polypeptide, carbohydrate, lipid, nucleic acid, etc.
  • a cellular binding ligand is a single chemical entity.
  • a cellular binding ligand is a complex of two or more discrete chemical entities associated with one another under relevant conditions by non-covalent interactions.
  • a cellular binding ligand may comprise a “generic” binding moiety (e.g., a class-specific antibody) and a “specific” binding moiety (e.g., an antibody or aptamers with a particular molecular target) that is linked to the partner of the generic biding moiety.
  • cellular binding ligands are or comprise polypeptides (including, e.g., antibodies or antibody fragments).
  • a cellular binding ligand comprises a recombinant antibody fragment (fAbs), a single chain variable fragment (scFv), and a single domain antibody (sdAb) fragment.
  • a cellular binding ligand comprises a recombinant antibody fragment (fAbs).
  • a cellular binding ligand comprises a single chain variable fragment (scFv).
  • a cellular binding ligand comprises a single domain antibody (sdAb) fragment.
  • Two cellular binding ligands can be linked to produce a bispecific immune cell engager (e.g., BiTE) or bispecific immune cell engager targeting ligand as described herein.
  • Chemotherapeutic agent As used herein, the term “chemotherapeutic agent” or “oncolytic therapeutic agent”(e.g., anti -cancer drug, e.g., anti-cancer therapy, e.g., immune cell therapy) has its art-understood meaning referring to one or more pro-apoptotic, cytostatic and/or cytotoxic agents, and/or hormonal agents, for example, specifically including agents utilized and/or recommended for use in treating one or more diseases, disorders or conditions associated with undesirable cell proliferation. In many embodiments, chemotherapeutic agents and/or oncolytic therapeutic agents are useful in the treatment of cancer.
  • a chemotherapeutic agent and/or oncolytic therapeutic agents may be or comprise one or more hormonal agents (e.g., androgen inhibitors), one or more alkylating agents, one or more anthracy clines, one or more cytoskeletal disruptors (e.g., microtubule targeting agents such as taxanes, maytansine, and analogs thereof), one or more epothilones, one or more histone deacetylase inhibitors HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and/or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and/or one or more analogs of one or more of the following (i.e., that share a relevant anti -
  • hormonal agents
  • a chemotherapeutic agent may be or comprise one or more of Actinomycin, all-trans retinoic acid, an Auiristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, curcumin,
  • Cytarabine Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine, and/or analogs thereof (e.g., DM1) Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof
  • a chemotherapeutic agent may be utilized in the context of an antibody-drug conjugate.
  • a chemotherapeutic agent is one found in an antibody-drug conjugate selected from the group consisting of: hLLl -doxorubicin hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLLl-SN-38, hRS7- Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20- Pro-2-P-Dox, hPAM4-Pro-2-pDox, hLLl -Pro-2 -P -Dox, P4/D1 O-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab ved
  • a chemotherapeutic agent may be or comprise one or more of famesyl-thiosalicylic acid (FTS), 4- (4- Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), estradiol (E2), tetramethoxystilbene (TMS), 8-tocatrienol, salinomycin, or curcumin.
  • FTS famesyl-thiosalicylic acid
  • CSH 4- (4- Chloro-2-methylphenoxy)-N-hydroxybutanamide
  • E2 estradiol
  • TMS tetramethoxystilbene
  • 8-tocatrienol salinomycin, or curcumin.
  • chemotherapeutic agents and/or oncolytic therapeutic agents for anti-cancer treatment comprise (e.g., are) biological agents such as tumor-infiltrating lymphocytes, CAR T-cells, antibodies, antigens, therapeutic vaccines (e.g., made from a patient’s own tumor cells or other substances such as antigens that are produced by certain tumors), immune-modulating agents (e.g., cytokines, e.g., immunomodulatory drugs or biological response modifiers), checkpoint inhibitors) or other immunologic/pharmacologic agents (e.g., PI3KD- selective inhibitor targeting myeloid cells or IPI-549).
  • biological agents such as tumor-infiltrating lymphocytes, CAR T-cells, antibodies, antigens, therapeutic vaccines (e.g., made from a patient’s own tumor cells or other substances such as antigens that are produced by certain tumors), immune-modulating agents (e.g., cytokines, e.g., immunomodulatory drugs or biological
  • immunologic agents include immunoglobins, immunostimulants (e.g., bacterial vaccines, colony stimulating factors, interferons, interleukins, therapeutic vaccines, vaccine combinations, viral vaccines) and/or immunosuppressive agents (e.g., calcineurin inhibitors, interleukin inhibitors, TNF alpha inhibitors).
  • immunostimulants e.g., bacterial vaccines, colony stimulating factors, interferons, interleukins, therapeutic vaccines, vaccine combinations, viral vaccines
  • immunosuppressive agents e.g., calcineurin inhibitors, interleukin inhibitors, TNF alpha inhibitors.
  • hormonal agents include agents for anti-androgen therapy (e.g., Ketoconazole, aBiraterone, TAK-700, TOK-oOl, Bicalutamide, Nilutamide, Flutamide, Enzalutamide, ARN-509).
  • Disease refers to cancer (e.g., solid tumors or metastatic tumors), brain injury (e.g., radiation, trauma), neurological disorders (e.g., Alzheimer’s disease), or inflammatory disorders (e.g., an inflammatory disease that affect the immune system, the pancreas, the blood sugar, and tissues in the body, e.g., an inflammatory disease that affects the nervous system, cardiovascular system, digestion system, integumentary system, musculoskeletal system, urinary system, reproductive system, endocrine system, or lymphatic system, e.g., diabetes).
  • cancer e.g., solid tumors or metastatic tumors
  • brain injury e.g., radiation, trauma
  • neurological disorders e.g., Alzheimer’s disease
  • inflammatory disorders e.g., an inflammatory disease that affect the immune system, the pancreas, the blood sugar, and tissues in the body, e.g., an inflammatory disease that affects the nervous system, cardiovascular system, digestion system, integumentary system
  • Marker refers to an entity or moiety whose presence or level is a characteristic of a particular state or event.
  • presence or level of a particular marker may be characteristic of presence or stage of a disease, disorder, or condition.
  • the term refers to a gene expression product that is characteristic of a particular immune cell type, immune cell subclass, activation of immune cells, and/or polarization of immune cells.
  • a presence or level of a particular marker correlates with activity (or activity level) of a particular signaling pathway, for example that may be characteristic of a particular class of immune cells.
  • 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.
  • composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
  • active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • 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 or Radioisotope 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.
  • a radiolabel is one used in positron emission tomography (PET).
  • a radiolabel is one used in singlephoton emission computed tomography (SPECT).
  • radioisotopes comprise " m Tc, m In, 64 Cu, 67 Ga, 186 Re, 188 Re, 153 Sm, 177 Lu, 67 Cu, 123 1, 124 1, 125 I, n C, 43 N, 15 0 , 18 F, 186 Re, 188 Re, 153 Sm, 161 HO, 177 LU, 149 Pm, 90 Y, 213 Bi, 103 Pd, 103 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 67 Cu, 105 Rh, in Ag, 89 Zr, 225 Ac, 192 Ir, and 89 Zr.
  • the radioisotope is not attached to the described nanoparticle conjugates. In certain embodiments, the radioisotope is attached to a second nanoparticle. In certain embodiments, the radioisotope (e.g., 89 Zr) is attached the described nanoparticle conjugates.
  • a therapeutic radioisotope can promote cytotoxic responses that may be additive/synergize with those of the described nanoparticles itself (e.g., a base particle having no ligands attached thereto) and/or the described nanoparticle conjugates.
  • 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.
  • Therapeutically effective amount is meant an amount that produces the desired effect for which it is administered.
  • the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition.
  • a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
  • therapeutically effective amount does not in fact require successful treatment be achieved in a particular individual.
  • a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.
  • reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.).
  • tissue e.g., a tissue affected by the disease, disorder or condition
  • fluids e.g., blood, saliva, serum, sweat, tears, urine, etc.
  • a therapeutically effective amount of a particular agent or therapy may be formulated and/or administered in a single dose.
  • a therapeutically effective agent may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.
  • Therapeutic agent in general refers to any agent that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect when administered to a subject.
  • 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.
  • Tumov refers to an abnormal growth of cells or tissue.
  • a tumor may comprise cells that are precancerous (e.g., benign), malignant, pre- metastatic, metastatic, and/or non-metastatic.
  • a tumor is associated with, or is a manifestation of, a cancer.
  • a tumor may be a solid tumor.
  • 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.
  • Bispecific immune cell engagers e.g., Bispecific T cell Engagers ("BiTEs")
  • BiTEs Bispecific T cell Engagers
  • the bispecific immune cell engager engages immune cells and/or heterogeneous tumor cells, depending on the design of the BiTE, at the site of the diseased cell.
  • Bispecific immune cell engagers comprise two cellular binding moieties [e.g., an immunomodulatory/stimulatory ligands, e.g., single-chain variable fragments (scFv)] attached by a linker.
  • a first cellular binding moiety may bind to an immune cell-specific molecule, e.g., CD3, and a second cellular binding moiety may bind to a disease-associated antigen (e.g., a tumor-associated antigen, e.g., PSMA, e.g., an inflammatory-associated antigen, e.g., brain injury-associated antigen, a neurological disease-associated antigen).
  • a disease-associated antigen e.g., a tumor-associated antigen, e.g., PSMA, e.g., an inflammatory-associated antigen, e.g., brain injury-associated antigen, a neurological disease-associated antigen.
  • Bispecific immune cell engagers can be specifically developed to target disease-associated antigens associated with a variety of primary and metastatic liquid (i.e., hematological) and solid tumors and other pathological states, such as brain injury (e.g., radiation, trauma) and neurological disorders.
  • An example of an immune cell-specific molecule that can be targeted with bispecific immune cell engagers include Toll receptors (e.g., TLR9), a pattern recognition receptor (PRR).
  • PRRs are a class of receptors that bridge innate and adaptive immunity and serve as critical co-stimulatory molecules of immune cells, notably myeloid cells (macrophages and dendritic cells).
  • PRRs serve as sensors that can recognize pathogen-associated molecules (PAMPs) or 'external' danger signals, such as viral DNA, but can also be recruited and activated upon recognition of endogenous stress signals, i.e., damage-associated molecular patterns (i.e., DAMPs).
  • PAMPs pathogen-associated molecules
  • DAMPs damage-associated molecular patterns
  • DAMPs may be secreted from apoptotic host cells or released from damaged senescent cells (e.g., extracellular ATP, mitochondrial DNA, heat shock protein). DAMPs may bind to PRRs of microglia and other immune cells leading to the stimulation of inflammatory cascades by activating different pro-inflammatory transcription factors (e.g., nuclear factor kappa B or NF-KB).
  • PRRs include Toll-like receptors (TLRs).
  • Natural endo/exogenous or synthetic PRR agonists such as synthetic oligodeoxynucleotides (ODNs) containing the unmethylated cytosine-phosphate-guanine (CpG) motif serve as potent pro- inflammatory stimulants of the innate immune system.
  • CpG motifs bind to Toll-like receptor 9 (TLR9) in the endosome of antigen presenting cells (APCs), promoting expression of co-stimulatory molecules, secretion of inflammatory cytokines, and development of CD8+ T cell responses.
  • TLR9 Toll-like receptor 9
  • APCs antigen presenting cells
  • these PRRs agonists have also been shown to overcome resistance to T-cell targeted immune checkpoints anti-CTLA-4 and anti-PD-l/PD-Ll .
  • the multi- therapeutic activities of the described immunomodulatory nanoparticle conjugates may improve efficacy in poorly immunogenic models, while also limiting resistance.
  • Examples of disease cell-specific molecules that can be targeted with immune cell engager targeting ligands include a tumor-associated antigen, e.g., PSMA, e.g., an inflammatory-associated antigen, e.g., brain injury-associated antigen, a neurological disease- associated antigen, or immune cell populations.
  • a tumor-associated antigen e.g., PSMA
  • an inflammatory-associated antigen e.g., brain injury-associated antigen, a neurological disease- associated antigen, or immune cell populations.
  • the present disclosure provides for engineered immunotherapies and immunomodulatory particle compositions comprised of CAR-bispecific immune cell engager engineered immune cells and silica nanoparticles, respectively, as described herein.
  • the described base nanoparticles themselves exhibit significant intrinsic cytotoxic and proinflammatory activities (e.g., activities that modulate macrophage phenotype, e.g., via augmentation of interferons that stimulate the immune system) within the tumor microenvironment.
  • immune cell engager targeting ligands that are comprised of immunomodulatory/stimulatory ligands (e.g., adjuvants), and used in conjunction with other immunotherapies, such as immune checkpoint blockade (ICB) therapeutics and other immune-targeting inhibitors.
  • immunomodulatory/stimulatory ligands e.g., adjuvants
  • other immunotherapies such as immune checkpoint blockade (ICB) therapeutics and other immune-targeting inhibitors.
  • the nanoparticles comprise ultrasmall nanoparticles or “C or C’ dots,” which are fluorescent, organo-silica core shell particles that have diameters controllable down to the sub-10 nm range with a range of modular functionalities.
  • C or C’ dots are described by U.S. Patent No. 8298677 B2 “Fluorescent silica-based nanoparticles”, U.S. Publication No. 2013/0039848 Al “Fluorescent silica-based nanoparticles”, and U.S. Publication No. US 2014/0248210 Al “Multimodal silica-based nanoparticles”, the contents of which are incorporated herein by reference in their entireties.
  • nanoparticles are described by WO2022221693A1 “Aluminosilicate nanoparticle sensors and uses thereof’, WO2022187261A1 “Templated materials, methods of making same, and uses thereof’, WO2021092065 Al “Ultrasmall nanoparticles and methods of making, using and analyzing the same”, W02021101704A2 “Asymmetric porous materials, methods of making same, and uses thereof’, WO2021087485A1 “Light patterning of silica nanocage materials”, WO2020214741A1 “Functionalized silica nanorings, methods of making same, and uses thereof’, WO2019213456A1 “Ultrasmall nanoparticles and methods of making, using and analyzing the same”, WO2019195858A1 “Inorganic nanocages, and methods of making and using same”, the contents of which are incorporated herein by reference in their entireties.
  • Incorporated into the silica matrix of the core are near-infrared dye molecules, such as Cy5.5, which provides its distinct optical properties.
  • Surrounding the core is a layer or shell of silica.
  • the silica surface is covalently modified with silyl-polyethylene glycol (PEG) groups to enhance stability in aqueous and biologically relevant conditions.
  • PEG silyl-polyethylene glycol
  • These particles have been evaluated in vivo and exhibit excellent clearance properties owing largely to their size and inert surface.
  • PEG silyl-polyethylene glycol
  • C or C’ dots are chemical sensing, non-optical (PET) image contrast and in vitrolin vivo targeting capabilities, which enable their use in visualizing lymph nodes for surgical applications, and melanoma detection in cancer.
  • C or C’ dots provide a unique platform for drug delivery due to their physical properties as well as demonstrated human in vivo characteristics.
  • These nanoparticle conjugates are ultrasmall (e.g., sub-8 nm) and benefit from targeted delivery (e.g., TLR-9) and EPR effects in tumor microenvironments, while retaining desired clearance and pharmacokinetic properties.
  • bispecific immune cell engagers e.g., BiTEs
  • nanoparticles described herein e.g., silica nanoparticles, e.g., C dots or other nanoparticles
  • the described nanoparticles do not have bispecific immune cell engagers targeting ligands (e.g., BiTE targeting ligands) conjugated thereto.
  • drug constructs are covalently attached to nanoparticles or described herein.
  • Nanoparticles for drug delivery provide good biostability, minimize premature drug release, and exhibit controlled release of the bioactive compound.
  • peptide- based linkers are used for the described nanoparticle conjugates and other applications described herein. These linkers, in the context of antibodies and polymers, are stable both in vitro and in vivo, with highly predictable release kinetics that rely on enzyme catalyzed hydrolysis by lysosomal proteases.
  • cathepsin B a highly expressed protease in lysosomes, can be utilized to facilitate drug release from macromolecules. By incorporating a short, protease sensitive peptide between the macromolecular backbone and the drug molecule, controlled release of the drug can be obtained in the presence of the enzyme.
  • the described nanoparticles exhibit intrinsic therapeutic capabilities that (1) modulate the tumor microenvironment (TME) toward a pro-inflammatory phenotype, (2) increase immune cell activation and cytotoxicity in the TME, and (3) target cancer cells directly for cell death through the mechanism of ferroptosis.
  • the described nanoparticles may not have a plurality of bispecific immune cell engagers targeting ligands (e.g., BiTE targeting ligands) attached thereto.
  • the nanoparticle comprises 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.
  • the nanoparticles may comprise one or more polymers, e.g., one or more polymers that have been approved for use in humans by the U.S. Food and Drug Administration (FDA) under 21 C.F.R. ⁇ 177.2600, including, but not limited to, polyesters (e.g., polylactic acid, poly(lactic-co- gly colic acid), polycaprolactone, polyvalerolactone, poly(l,3-dioxan-2-one)); polyanhydrides (e.g., poly(sebacic anhydride)); poly ethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; polycyanoacrylates; copolymers of PEG and polyethylene oxide) (PEG).
  • the diameter of the immunomodulatory nanoparticle conjugates described herein is not substantially increased by the one or more polymers.
  • the nanoparticles may comprise one or more degradable polymers, for example, certain polyesters, polyanhydrides, polyorthoesters, polyphosphazenes, polyphosphoesters, certain polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, poly(amino acids), polyacetals, polyethers, biodegradable polycyanoacrylates, biodegradable polyurethanes and polysaccharides.
  • degradable polymers for example, certain polyesters, polyanhydrides, polyorthoesters, polyphosphazenes, polyphosphoesters, certain polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, poly(amino acids), polyacetals, polyethers, biodegradable polycyanoacrylates, biodegradable polyurethanes and polysaccharides.
  • biodegradable polymers that may be used include but are not limited to polylysine, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), poly(lactide-co-caprolactone) (PLC), and poly(glycolide-co- caprolactone) (PGC).
  • PLA poly(lactic acid)
  • PGA poly(glycolic acid)
  • PCL poly(caprolactone)
  • PLG poly(lactide-co-glycolide)
  • PLA poly(lactide-co-caprolactone)
  • PLC poly(glycolide-co- caprolactone)
  • PLC poly(glycolide-co- caprolactone)
  • Another exemplary degradable polymer is poly (beta-amino esters), which may be suitable for use in accordance with the present application.
  • a nanoparticle can have or be modified to have one or more functional groups.
  • Such functional groups (within or on the surface of a nanoparticle) can be used for association with any agents (e.g., detectable entities, targeting entities, therapeutic entities, or PEG).
  • agents e.g., detectable entities, targeting entities, therapeutic entities, or PEG.
  • linkers e.g., (cleavable or (biodegradable) polymers such as, but not limited to, polyethylene glycol, polypropylene glycol, PLGA, etc.
  • a label for imaging and/or radiotherapy can be attached to the nanoparticle as described herein.
  • the nanoparticles comprises a therapeutic agent, e.g., a drug moiety (e.g., a chemotherapy drug) and/or a therapeutic radioisotope.
  • a therapeutic agent refers to any agent that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect, when administered to a subject.
  • the nanoparticles described herein demonstrate enhanced penetration of tumor tissue and diffusion within the tumor interstitium, e.g., for treatment of cancer, as described in International Patent Application No. PCT/US 17/30056 (“Compositions and Methods for Targeted Particle Penetration, Distribution, and Response in Malignant Brain Tumors,” filed April 28, 2016) by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety. Further described are methods of targeting tumor-associated macrophages, microglia, and/or other cells in a tumor microenvironment using such nanoparticles conjugates.
  • the nanoparticles described herein can be used to induce ferroptosis, as described in International Patent Application No. PCT/US16/34351 (“Methods of Treatment Using Ultrasmall Nanoparticles to Induce Cell Death of Cancer Cells via Ferroptosis,” filed on May 26, 2016) by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety.
  • the nanoparticles described herein can be used to induce ferroptosis, as described in International Patent Application No.
  • PCT/US18/63751 (“Methods of Cancer Treatment via Regulated Ferroptosis,” filed on December 4, 2018) by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety.
  • the nanoparticles described herein can be used to activate innate and adaptive immune responses within the tumor microenvironment, as described in International Patent Application No. PCT/US 19/66944 (“Inducing Favorable Effects on Tumor Microenvironment via Administration of Nanoparticle Compositions,” filed on December 17, 2019, by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety.
  • the nanoparticles described herein can be used to activate tumor cells and/or innate and adaptive immune responses within the tumor microenvironment, as described in International Patent Application No. PCT7US2022/034224 (“Nanoparticle-mediated Enhancement of Immunotherapy to Promote Ferroptosis-induced Cytotoxicity and Antitumor Immune Responses,” filed on June 21, 2022) by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety.
  • nanoparticle conjugates are described for treating targets in both the tumor and surrounding microenvironment, thereby enhancing efficacy of cancer treatment e.g., immunotherapies.
  • Multi -targeted kinase inhibitors and combinations of single-targeted kinase inhibitors have been developed to overcome therapeutic resistance.
  • multimodality combinations of targeted agents including particle-based compositions designed to carry small molecule inhibitors (SMIs), chemotherapeutics, radiotherapeutic labels, and/or immunotherapies can enhance treatment efficacy and/or improve treatment planning of malignant tumors, including malignant brain tumors. Coupled with molecular imaging labels, these vehicles permit monitoring of drug delivery, accumulation, and retention, which may, in turn, lead to optimal therapeutic indices.
  • SMSIs small molecule inhibitors
  • chemotherapeutics chemotherapeutics
  • radiotherapeutic labels radiotherapeutic labels
  • immunotherapies can enhance treatment efficacy and/or improve treatment planning of malignant tumors, including malignant brain tumors.
  • these vehicles coupled with molecular imaging labels, these vehicles permit monitoring of drug delivery, accumulation, and retention, which may, in turn, lead to optimal therapeutic indices.
  • radiolabels and/or fluorescent markers attached to (or incorporated in or on, or otherwise associated with) the nanoparticles provide quantitative assessment of 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 bispecific immune cell engagers targeting ligands (e.g., BiTE targeting ligands) to develop a drug delivery system with controlled pharmacological properties.
  • the described platforms determine the influence of targeting on 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 (Positron Emission Tomography) tracers are used as imaging agents.
  • PET tracers comprise 89 Zr, 64 Cu, [ 18 F] fluorodeoxyglucose.
  • the immunomodulatory nanoparticle conjugate includes these and/or other therapeutic radiolabels that may induce additional cytotoxic responses within the microenvironment.
  • the nanoparticle comprises one or more fluorophores.
  • Fluorophores comprise fluorochromes, fluorochrome quencher molecules, any organic or inorganic dyes, metal chelates, or any fluorescent enzyme substrates, including protease activatable enzyme substrates.
  • fluorophores comprise long chain carbophilic cyanines.
  • 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, Kodak X-SIGHT 751 (Carestream Health).
  • a multi- wavelength camera as described by Bradbury et al. US Publication No. US 2015/0182118 Al,
  • the imaging system used to image the lesion provides both static and functional assessments of the area of treatment (and its surroundings).
  • Example therapeutics and/or drugs that can be used include RTK inhibitors, such as dasatinib and gefitinib, can target either platelet-derived growth factor receptor (PDGFR) or EGFRmt+ expressed by primary tumor cells of human or murine origin (e.g., genetically engineered mouse models of high-grade glioma, neurospheres from human patient brain tumor explants) and/or tumor cell lines of non-neural origin.
  • Dasatinib and gefitinib analogs can be synthesized to enable covalent attachment to several linkers without perturbing the underlying chemical structure defining the active binding site.
  • checkpoint inhibitors can be used as therapeutics and/or drugs for treatment of disease such as cancer.
  • the surface chemistry, uniformity of coating (where there is a coating), surface charge, composition, concentration, frequency of administration, shape, and/or size of the nanoparticle conjugates can be adjusted to produce a desired therapeutic effect.
  • the nanoparticle conjugate comprises a chelator, for example, 1,4,8, 1 l-tetraazabicyclo[6.6.2]hexadecane-4,l 1- diyl)diacetic acid (CB-TE2A); desferoxamine (DFO); diethylenetriaminepentaacetic acid (DTPA); 1,4,7, 10-tetraazacyclotetradecane- 1,4,7, 10-tetraacetic acid (DOTA); thylenediaminetetraacetic acid (EDTA); ethylene glycolbis(2-aminoethyl)-N,N,N',N'- tetraacetic acid (EGTA); 1,4,8, 1 l-tetraazacyclotetradecane-1,4,8,1 1-tetraacetic acid (TETA); ethylenebis-(2-4 hydroxy-phenylglycine) (EHPG); 5-C1-EHPG; 5Br-EHPG; 5- Me-EH
  • EHPG ethylenebis
  • the nanoparticle conjugate comprises an azide moiety.
  • an azide moiety is attached to an antibody fragment for conjugation to a nanoparticle described herein.
  • azide moieties are attached to bispecific immune cell engagers targeting ligands (e.g., BiTE targeting ligands) (e.g., CD3/TYPR1 scFv, e.g., PSMA/CD3, e.g., CD8/ TYPR1), for instance, for conjugation to a nanoparticle described herein.
  • BiTE targeting ligands e.g., CD3/TYPR1 scFv, e.g., PSMA/CD3, e.g., CD8/ TYPR1
  • the nanoparticle conjugate comprises more than one chelator.
  • the radioisotope-chelator pair is 89 Zr-DFO. In certain embodiments the radioisotope-chelator pair is 177 Lu-DOTA. In certain embodiments, the radioisotopechelator pair is 225 Ac-DOTA.
  • the nanoparticle conjugate may be associated with PET labels and/or optical probes. Nanoparticle conjugates may be observed in vivo (e.g., via PET) to evaluate drug accumulation in a target site. For example, nanoparticle conjugates with PET labels (e.g., without drug substances) may be administered first. Then, by analyzing the in vivo PET images of the nanoparticles, drug (e.g., conjugated with nanoparticle conjugates) concentration and accumulation rate in the tumor may be estimated. The dose may be determined based on the obtained estimation to provide personalized medicine (e.g., tumor size rather than the patient’s body weight). In some embodiments, a radiolabeled drug may be traced in vivo.
  • nanoparticle conjugates with optical probes may be used for intraoperative imaging (e.g., where surface of tissue/tumor is exposed) and/or biopsies of tumors.
  • a therapeutically effective amount of the described immunomodulatory nanoparticle conjugates is used is compositions administered to a subject herein.
  • a person of skill in the art would be able to select an appropriate dosage for administration based on administration route, cancer, disease, condition, and/or subject being treated, among other factors.
  • concentrations of up to 15 pM (e.g., from about lOOnM to about 15 pM) of immunomodulatory nanoparticle conjugates are administered to a subject.
  • a therapeutically effective amount of nanoparticle conjugates are administered to a subject herein to modulate a tumor microenvironment of the subject (e.g., via “switching” on or off the tumor microenvironment, e.g., via “switching” on or off the regulatory T cell (Treg) population in the tumor microenvironment).
  • a therapeutically effective amount of nanoparticle conjugates are administered at and/or near a site of the cancer (e.g., into a tumor), disease, and/or condition.
  • a high local concentration of nanoparticle conjugates is administered to a subject at a site of the cancer, disease, and/or condition.
  • a high local concentration is a local concentration within a range from 0.18 pM to 1.8 pM in cancer cells and/or tumor tissue and/or diseased tissue of a subject.
  • a high concentration is a local concentration in cancer cells and/or tumor tissue and/or diseased tissue of at least 0.18 pM, at least 0. 3 pM, at least 0.4 pM, at least 0.5 pM, at least 0.6 pM, or at least 1 pM.
  • a therapeutically effective local concentration is dependent on tumor type and/or disease type and/or condition type and/or subject.
  • a high local concentration is a local concentration within a range from 1 pM to 15 pM in cancer cells and/or tumor tissue and/or diseased tissue of a subject.
  • a high concentration is a local concentration in cancer cells and/or tumor tissue and/or diseased tissue of at least 1 pM, at least 2 pM, at least 3 pM, at least 4 pM, at least 5 pM, at least 6 pM, at least 7 pM, at least 8 pM, at least 9 pM, at least 10 pM, at least 11 pM, at least 12 pM, at least 13 pM, or at least 14 pM.
  • a therapeutically effective local concentration is dependent on tumor type and/or subject.
  • immunomodulatory nanoparticle conjugates can be administered as a single dosage (e.g., as a bolus injection).
  • nanoparticle conjugates can be administered in two, three, four, five, six, seven, eight, or more dosages.
  • nanoparticle conjugates can be administered along with (e.g., simultaneously with) engineered immune cells (e.g., a macrophage, e.g., a neutrophil, e.g., a T cell (e.g., a Steapl CAR-T cell), e.g., an NK cell).
  • engineered immune cells e.g., a macrophage, e.g., a neutrophil, e.g., a T cell (e.g., a Steapl CAR-T cell), e.g., an NK cell.
  • nanoparticle conjugates can be administered prior to administration of engineered immune cells. In some embodiments, (e.g., a therapeutically effective amount of) nanoparticle conjugates can be administered after administration of engineered immune cells. In some embodiments, (e.g., a therapeutically effective amount of) nanoparticle conjugates (e.g., the same nanoparticle conjugates, different nanoparticle conjugates) can be administered before and after administration of engineered immune cells.
  • CAR immune cells engineered with bispecific immune cell engagers in combination with nanoparticles for localized target-specific therapy
  • compositions comprising (1) engineered immune cells that have been engineered to express a chimeric antigen receptor (CAR) and release a bispecific immune cell engager (e.g., BiTE) (CAR.BiTE engineered immune cell), and (2) ultrasmall nanoparticles as described herein.
  • CAR chimeric antigen receptor
  • BiTE bispecific immune cell engager
  • CAR-bi specific immune cell engager e.g., CAR.BiTE
  • ultrasmall immunomodulatory nanoparticles can stimulate a subject's immune response to improve disease burden without significant off-target effects by directing immune cells to disease-specific sites, e.g., targeted therapy of primary and metastatic tumors and other diseases (e.g., inflammatory, neurodegenerative), as described herein.
  • nanoparticles have BiTE targeting ligands attached thereto. In some embodiments, the nanoparticles do not have BiTE targeting ligands attached thereto.
  • the present disclosure provides an exemplary protocol to produce CAR.BiTE engineered immune cells:
  • Packaging cells also express the CAR.BiTE engineered immune cells.
  • Infect immune cells of choice e.g., CAR.BiTE engineered immune cells can be tested in vitro in 2-D or 3-D models, including organoids.
  • CAR.bispecific immune cell engager e.g., CAR.BiTE engineered immune cells comprise a synthetic “on/off” system (e.g., a synthetic NOTCH system).
  • a synthetic NOTCH receptor that recognizes a specific priming antigen, such as the heterogeneous but tumor-specific glioblastoma neoantigen epidermal growth factor receptor splice variant III (EGFRvIII) or the central nervous system (CNS) tissue-specific antigen myelin oligodendrocyte glycoprotein (MOG) can be used to locally induce expression of a CAR. This provides thorough but controlled tumor cell killing by targeting antigens that are homogeneous but not absolutely tumor specific.
  • a synthetic “on/of ’ switch system that regulates CAR expression averts tonic signaling and exhaustion, maintaining a higher fraction of the T cells in a naive/stem cell memory state. See, e.g., Choe et al., Sci Transl Med. 2021 April 28; 13(591): doi: 10.1126/scitranslmed.abe7378, the contents of which is hereby incorporated by reference herein in its entirety.
  • the present disclosure describes immunomodulatory nanoparticle conjugates comprising a nanoparticle and a plurality of immune cell engager targeting ligands (comprised of at least two species) (e.g., BiTE targeting ligands, e.g., dual targeting ligands) conjugated to the nanoparticle.
  • Immune cell engager targeting ligands can be selected and engineered based on the type of indication being pursued.
  • immune cell engager targeting ligands can be ligands targeting antigens expressed in tumor cells, anti-inflammatory peptides useful for treatment of neurological disorders or brain injury (e.g., radiation, trauma), or immune cells.
  • bispecific immune cell engagers include CD3/TYPRlor PSMA/CD3 or CD8/TYPR1.
  • Bispecific immune cell engagers can be bispecific T-cell engagers, bispecific macrophage engagers, bispecific neutrophil engagers, and the like.
  • immune cell engager targeting ligands are associated with each other via a linker (e.g., as in a BiTE) (e.g., a flexible linker (e.g., a glycine-serine linker), a cleavable linker (e.g., enzymatically cleavable linker)).
  • a linker e.g., as in a BiTE
  • a linker e.g., a flexible linker (e.g., a glycine-serine linker), a cleavable linker (e.g., enzymatically cleavable linker)
  • immune cell engager targeting ligands are not associated with each other (e.g., are not associated via a linker, e.g., dual targeting ligands).
  • a composite targeting ligand can be comprised of an immunomodulatory/ stimulatory ligand (e.g., a TLR agonist (e.g., an oligodeoxynucleotides (ODNs)) containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs))) and a single chain fragment (e.g., FcyRII/CD32 present on antigen-presenting cells) that can be conjugated to a nanoparticle.
  • a TLR agonist e.g., an oligodeoxynucleotides (ODNs)
  • ODNs unmethylated cytosine-phosphate-guanine
  • FcyRII/CD32 present on antigen-presenting cells
  • the described base nanoparticles themselves exhibit significant intrinsic cytotoxic and proinflammatory activities (e.g., activities that modulate macrophage phenotype, e.g., via augmentation of interferons that stimulate the immune system) within the tumor microenvironment.
  • cytotoxic and proinflammatory activities e.g., activities that modulate macrophage phenotype, e.g., via augmentation of interferons that stimulate the immune system
  • responses can be further enhanced by surface attachment of immune cell engager targeting ligands (e.g., BiTE targeting ligands, e.g., dual targeting ligands) that have an immunomodulatory/stimulatory ligand (e.g., proinflammatory ligands), and used in conjunction with immune checkpoint blockade (ICB) therapeutics and other experimental immunotherapies.
  • IRB immune checkpoint blockade
  • systemic administration of the described nanoparticle with or without immune cell engager targeting ligands e.g., BiTE targeting ligands, e.g., dual targeting ligands
  • immune cell engager targeting ligands e.g., BiTE targeting ligands, e.g., dual targeting ligands
  • PRRs multiple pattern recognition receptors or PRRs, such as TLR-, and IFN-related pathways
  • the described base nanoparticle itself shows enhanced immunologic responses (e.g., affecting upregulation of the RIG/MDA5-MAVS and cGAS- STING pathways) in specific melanoma models treated systemically.
  • such effects can be enhanced by attachment of a plurality of immune cell engager targeting ligands (e.g., BiTE targeting ligands, e.g., dual targeting ligands) to the base nanoparticle.
  • Other variables that can influence immunologic responses described herein include size of the nanoparticle, material composition of the nanoparticles, surface chemistry of the nanoparticle and particle topology, as described herein.
  • a probe can be a CD3/ TYPR1 scFv BiTE attached to a nanoparticle, where CpG targets T cells and TYPRlscFv targets cancer cells.
  • the nanoparticle itself, may also be (or have attached) another targeting moiety or drug.
  • a probe can be a CD3/ TYPR1 scFv targeting ligands (not a BiTE) attached to a nanoparticle, where CpG targets T cells and TYPRlscFv targets cancer cells.
  • the nanoparticle itself, may also be (or have attached) another targeting moiety or drug.
  • Other silica topologies or particle compositions may also be used.
  • immune cell engager targeting ligands e.g., BiTE targeting ligands, e.g., dual targeting ligands
  • immune cell engager targeting ligands comprise a plurality of cellular binding ligands as described herein.
  • the nanoparticles comprise from 1 to 100 discrete immune cell engager targeting ligands (e.g., of at least two different types) (e.g., BiTE targeting ligands, e.g., dual targeting ligands), wherein these ligands bind to receptors within/on tumor cells and/or within/on immune 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).
  • discrete immune cell engager targeting ligands e.g., of at least two different types
  • BiTE targeting ligands e.g., dual targeting ligands
  • these ligands bind to receptors within/on tumor cells and/or within/on immune cells (e.g., wherein the immunomodulatory nanoparticle conjugates have an average diameter no greater
  • the immunomodulatory nanoparticle conjugates further comprise a plurality of immunomodulatory/ stimulatory ligands (e.g., CpG).
  • the immunomodulatory nanoparticle conjugates comprise from 1 to 100 discrete immunomodulatory/stimulatory targeting ligands, e.g., from 1 to 30 discrete immunostimulatory ligands, e.g., from 1 to 20 discrete immunomodulatory/stimulatory ligands, e.g., from 1 to 10 discrete immunomodulatory/stimulatory targeting ligands.
  • the immunomodulatory nanoparticle conjugates comprise (e.g., has attached) one or more immune cell engager targeting ligands (e.g., BiTE targeting ligands, e.g., dual targeting ligands), e.g., for targeting cancer tissue/cells of interest.
  • immune cell engager targeting ligands e.g., BiTE targeting ligands, e.g., dual targeting ligands
  • the immunomodulatory nanoparticle conjugate comprises one or more targeting binding ligands (e.g., attached thereto), such as, but not limited to, small molecules (e.g., folates, dyes, etc.), aptamers (e.g., A10, AS 1411), polysaccharides, small biomolecules (e.g., folic acid, galactose, bisphosphonate, biotin), oligonucleotides, and/or proteins (e.g., (poly)peptides (e.g., aMSH, RGD, octreotide, AP peptide, epidermal growth factor, chlorotoxin, transferrin, etc.), antibodies, antibody fragments, proteins, etc.).
  • small molecules e.g., folates, dyes, etc.
  • aptamers e.g., A10, AS 1411
  • polysaccharides e.g., small biomolecules (e.g., foli
  • the immunomodulatory nanoparticle conjugate comprises one or more immune adjuvants (e.g., pattern recognition receptors, e.g., toll-like receptor agonists, e.g., antibody fragments) (and, optionally, a targeting agent).
  • the immunomodulatory nanoparticle conjugate comprises one or more contrast/imaging agents (e.g., fluorescent dyes, (chelated) radioisotopes (SPECT, PET), MR-active agents, CT-agents), and/or therapeutic agents (e.g., small molecule drugs, therapeutic (poly)peptides, therapeutic antibodies, (chelated) radioisotopes, etc.).
  • contrast/imaging agents e.g., fluorescent dyes, (chelated) radioisotopes (SPECT, PET), MR-active agents, CT-agents
  • therapeutic agents e.g., small molecule drugs, therapeutic (poly)peptides, therapeutic antibodies, (chelated) radioisotopes, etc.
  • nanoparticles comprising C’ dots are synthesized as discussed herein.
  • nanoparticles as discussed herein are characterized using a particle characterization technique (e.g., FCS, DLS, zeta-potential, UV-VIS absorption, emission spectroscopy, transmission electron microscopy).
  • a particle characterization technique e.g., FCS, DLS, zeta-potential, UV-VIS absorption, emission spectroscopy, transmission electron microscopy.
  • 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
  • nanoparticles with a ring or cage conformation may be selected for use in the engineered therapy.
  • hydrophobic “patchiness” of nanoparticles is controlled using the methods and techniques described herein.
  • the surface patchiness of the nanoparticles is used to control, among other things, tumor microenvironment response to nanoparticles.
  • two Cy5-maleimido dye derivatives with different net charges are used: negatively charged sulfo-Cy5(-)-maleimide dye (GE) or positively charged Cy5(+)-maleimide dye (Lumiprobe).
  • GE negatively charged sulfo-Cy5(-)-maleimide dye
  • Luiprobe positively charged Cy5(+)-maleimide dye
  • 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
  • the synthesis of the silica core of the nanoparticle is controlled as described herein.
  • the water-based synthesis of C’ dots enables control of the silica core size at the level of a single atomic SiCh layer.
  • the exceptional degree of particle size control allows generation of nanoparticles (e.g., C’ dots) with overall particle size maintained below the cut-off for renal clearance (e.g., below 15nm) to reduce unwanted off-target accumulations (e.g., in the liver), while varying sizes of core and/or shell.
  • Silica core size is reduced by increasing reaction temperature and/or by decreasing the time of core growth before PEG-silane is added.
  • the length of the PEG-silane chains is increased to maintain an overall hydrodynamic size of the nanoparticle.
  • Changing relative sizes of silica core and/or PEG shell of otherwise same hydrodynamic size of nanoparticles allows decoupling contributions of silica core and PEG shell to ferroptosis and/or immune cell priming.
  • addition of compounds (e.g., aluminum-containing compounds) to the nanoparticle core can boost activation of cell populations within the tumor microenvironment.
  • the silica core composition of nanoparticles is modulated as described herein.
  • modulation of the composition of the silica cores affects affinity of iron to C’ dots, which can be chelated by silanol (-SiOH) surface groups in micropores of the sol-gel derived silica core.
  • Silica core composition can be varied, e.g., by the addition of aluminum sec-butoxide, mercapto-silane, and/or iodo-silane moieties into the aqueous sol-gel reaction mixture.
  • 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) I 3 -(tri hydroxy si lyljpropyl 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 tri hydroxy si lyljpropyl 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 pM 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.
  • the present disclosure describes bispecific immune cell engagers (e.g., BiTEs) that comprise a first cellular binding moiety that targets tumor antigens or receptors (including TLR), as well as an immune target.
  • the first cellular binding moiety comprises an agonist targeting a class of receptors termed pattern recognition receptors (PRR).
  • PRRs are innate immune system activators, such as Toll-like receptors (TLRs), cyclic GMP-AMP synthase (cGAS)- stimulator of interferon genes (STING), and retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), among others.
  • PRRs can be activated by pathogens (e.g., viral DNA) or by endogenous damage-associated molecular patterns (or DAMPS) synthesized and/or released upon cellular stress, apoptosis, or necrosis (e.g., immune-related cell death).
  • DAMPs emitted during this process include heat-shock proteins (HSPs), ATP released from dying cells into the extracellular space, calreticulin, and type I interferons (IFNs).
  • HSPs heat-shock proteins
  • IFNs type I interferons
  • PRRs Upon binding their respective endo/exog enous or synthetic agonists, PRRs serve as critical costimulatory molecules for immune cells, notably myeloid cells (macrophages and dendritic cells), promoting pro-inflammatory gene expression, activation of phagocytosis, and antigen presentation in the TME, and exerting effects that bridge non-specific and specific immunity.
  • immune cells notably myeloid cells (macrophages and dendritic cells)
  • a PRR agonist is or comprises an unmethylated cytosine- phosphate-guanine oligodeoxynucleotide (CpG-ODN).
  • CAR.BiTE engineered immune cells can serve as a PRR agonist for improving therapeutic efficacy via its broad-ranging modulation of multiple signaling pathways: STING-, TLR-, RIG-I, and interferon (IFN).
  • Engineered immunotherapies for targeting suppressive myeloid cells and limiting resistance [0179] Despite their promise as therapeutics, small molecule immunomodulators, such as CpG, and agents producing inflammatory responses within the TME (e.g., peptides, drugs) usually demonstrate suboptimal PK and are prone to degradation. Moreover, such agents often exhibit nonspecific interactions with proteases, nucleases and immune cells, which can diminish their immunomodulatory activities and lead to adverse reactions compromising their safety.
  • NPs biocompatible nanoparticles
  • TLR Toll-like receptor
  • smaller core particle sizes e.g., 15 nm were found to improve the immunostimulatory properties of CpG-conjugated gold NPs, over larger size particles (greater than 30 nm) and exhibited higher specificity for targeting immune receptors.
  • the present disclosure provides for engineered immunotherapies and immunomodulatory particle compositions comprised of engineered immune cells and targeted silica nanoparticles for localized targeted therapy in the treatment of cancer and other diseases, injuries, or conditions.
  • compositions and methods for identifying, evaluating, and evaluating candidate therapies detail compositions and methods for identifying, evaluating, and evaluating candidate therapies.
  • Candidate immunotherapies can be evaluated and compared at each stage of translational development based on key selection criteria and discrete endpoints - illustrated as a series of screening steps (boxes) as shown in FIG. 1.
  • a clinically translatable lead bispecific scFv-C’ dot whose combined anti-tumor cytotoxic and immunomodulatory properties can be leveraged to reverse immune suppression and synergize with TA99-CAR T cells to prolong survival over that with singleagent therapies in AM models without on-target/off-tumor toxicity.
  • the transgenic AM model used for this work better recapitulates human AM cancer biology and is therefore ideal for product translation.
  • contributions to overall treatment responses include TA99-CAR T cells and particle-driven T-cell-based IT; the latter including the self-therapeutic C’ dot properties.
  • criteria are met (see FIG. 1), specifically survival benefit without on-target/off-tumor toxicity and (CD8+/Treg, a lead bispecific scFv-C’ dot can be advanced to the clinic for a Phase 1 trial to establish safety and dosing regimes.
  • treatment paradigms described herein combining T-cell based particle immunotherapies with CAR T cells can impart significant and clinically promising multi-therapeutic benefits to the treatment of AM models via enhanced immunogenic and antitumor cytotoxic responses, improving therapeutic outcomes and toxicologic profiles over those found with single-agent therapies.
  • the present Experimental Embodiments includes three stages of development.
  • Stage I bispecific scFv-C’ dots are developed by conjugating a base particle with varying numbers of bispecific anti-TYRPl/CD3 T cell engagers (BiTEs) or dual -targeting anti-TYRPl- and -CD3-scFvs, and their structural, biological, and immunostimulatory properties assessed in tumor and immune cell populations relative to controls.
  • BiTEs bispecific anti-TYRPl/CD3 T cell engagers
  • dual -targeting anti-TYRPl- and -CD3-scFvs dual -targeting anti-TYRPl- and -CD3-scFvs
  • Stage II bispecific scFv-C’ dot candidates undergo screening pharmacokinetic (PK) studies in subcutaneous (SQ) humanized AM xenograft models after zirconium-89 ( 89 Zr) radiolabeling to identify probes exhibiting favorable targeted uptake and clearance kinetics. Lead candidates can then be combined with TA99-CAR T cells and optimal dose-schedules and sequencing evaluated to achieve maximize efficacy, reduce adverse events, and identify synergistic combination strategies.
  • Stage III selected combinatorial strategies used in Stage II are evaluated in a GEM model to investigate the extent to which the TME influences efficacy, immune suppression/escape, and T cell effector function. Contributions to overall responses from particle- driven cytotoxicity, immune cell activity, and pro-inflammatory responses can be determined. Each stage is further detailed below. I. Identification of C’dots conjugated with BiTEs and/or scFvs
  • the present example describes identification and optimization of structural properties of C’ dots, conjugated with BiTEs or individual scFvs, that maximally enhance immunogenicity, immune cell function, and cytotoxic responses in AM cell lines.
  • Pilot data indicates that base nanoparticles as described herein possess multiple intrinsic therapeutic properties.
  • modification of specific structural parameters, such as particle surface composition and ligand density can enhance the specificity and potency of target engagement, as well as other biological activities.
  • modifying the surface of C’ dots with variable numbers of synthetically engineered anti-TYRPl, CD3, and GITR scFvs - either as a BiTE or individually with separate scFvs - may enhance target-specific engagement, binding affinity, tumor cell lysis, and/or T-cell functionality.
  • TYRP1 -targeting -CAR T (or TA99-CAR T) cells can be used for in vitro human AM and murine CM co-culture assays, as these cells have demonstrated antigen-specific activation and cytotoxic activity against both human and murine CM cells.
  • variable light and heavy regions of human anti-TYRPl antibodies as derived from the SAbDab database (the Structural Antibody Database: https://opig.stats.ox.ac.uk/webapps/sabdab-sabpred/sabdab) can be used for the present experimental studies.
  • Both variable light and heavy regions of human and mouse CD3 (for Stage III) or GITR antibodies can be used.
  • CD3 and GITR can be used interchangeably with TYRP1.
  • Genetic sequences (gBlocks) can be constructed using a codon optimization tool (Integrated DNA Technologies, Newark, NJ).
  • Linearized gBlocks can be cloned into lentiviral vectors for transduction of Lenti-X cells (TakaraBio) and synthesis/secretion of scFvs.
  • Vectors co-express a reporter protein for assessing transduction efficiency and for enriching positively transduced cell populations.
  • cells can be cultured, and supernatant processed to obtain purified His- tagged proteins (e.g., either anti-TYRPl/CD3 (or anti-TYRPl/GITR) BiTEs (referred to in the Experimental section as BiTEs) or individual TYRP1, CD3, (or GITR) scFvs (dual -targeting construct). Characterization of the size, sequence, binding specificity, and stability of these constructs can be performed using preparative/analytic size-exclusion chromatography, western blot, mass spectroscopy, and surface plasmon resonance. A person of skill in the art would understand, based on the present disclosure, appropriate methods to characterize constructs.
  • bispecific scFv-C’ dots with varying ligand density and non-targeted controls (C’ dots).
  • BiTE-targeting or bispecific TYRP1- and CD3- (and GITR)-targeting C’ dots the latter designated TYRP1/CD3 (or TYRP1/GITR)-C’ dots, along with non-targeting C’ dot controls and deferoxamine- (DFO-) chelator modified C’ dots, can be synthesized, purified, and characterized following well-established protocols and advanced characterization techniques. Characterization can utilize high-performance liquid chromatography (HPLC) methods for facilitating the clinical translation of final C’ dot products.
  • HPLC high-performance liquid chromatography
  • DFO and BiTEs can be conjugated to C’ dots using post-PEGylation surface modification by insertion (PPSMI) methods employing validated protocols from earlier scFv ligand studies.
  • PPSMI post-PEGylation surface modification by insertion
  • ATMS Aminopropyl- trimethoxy-silane
  • Resulting NEE-C’ dots can first be reacted with l-(4-isothiocyanatophenyl) containing p-SCN-Bn-DFO.
  • DBCO Dibenzocyclooctyne
  • DBC0-PEG4-N- hydroxysuccinimidyl ester DBC0-PEG4-N- hydroxysuccinimidyl ester
  • Azide-bearing scFvs can then efficiently be “clicked” onto the particles via azide-alkyne cycloaddition in two batches: either as a single BiTE or as separate scFvs.
  • the number of DFO chelators, DBCOs, and scFvs can be controlled by the reaction concentration ratios of ligands to particles (i.e., 1-2 DFOs, 20-30 DBCOs, and 1-3 BiTEs or scFvs per particle), with particle concentrations derived from fluorescence correlation spectroscopy (FCS).
  • the number of DFOs, DBCOs, or bispecific moieties per particle can be quantified by UV-Vis spectral deconvolution methods by using Cy5 particle fluorescence and FCS-derived particle concentrations.
  • Screening criteria used are as follows: (i) scFv, BiTE, C’ dot purity > 95%; (ii) C’ dot hydrodynamic diameter ⁇ 8 nm; (iii) number of Cy5 dyes (>1); (iv) TYRP1/CD3, TYRP1/GITR BiTEs, anti-TYRPl/-CD3 scFvs, anti-TYRPl/-GITR scFvs per C’ dot: 1- 3; (v) DFO ligands per C’ dot: 2 - 5.
  • Patient-derived AM cell lines SK-Mel709B (TYRPl high ), SK-MellO94A (TYRPl high ), SK-Mel990A (TYRP intermediate ), exhibiting a spectrum of TYRP1 expression (TYRP1+) , and a TYRP1- (control) line, B78Hl-TYRPlnull/B16, can be used for all assays.
  • a TYRPl hlgh murine CM cell line, B16-F10 can be used for comparative purposes.
  • Human AM cells and murine CM cells can be transduced with a retrovirus encoding a firefly luciferase Luc) construct.
  • leukopaks can be obtained from the New York Blood Center. Cells can be isolated using a CD3 negative selection kit (STEMCell). T cells can be cultured in RPMI 1640 supplemented with 100 lU/mL interleukin-2 (Proluekin, Novartis). All media can be supplemented with 10% heat-inactivated FBS, 2 mmol/L L-glutamine (Invitrogen), 100 units/mL penicillin, and 100 mg/mL streptomycin (Invitrogen) and stimulated with CD3/CD28 beads (Invitrogen) per established protocols. An EasySepTM Human CD+CD1271owCD25+ Regulatory T cell Isolation Kit can be used for Treg isolation.
  • IC50 measurements of bi specific C’ dot constructs specific for TYRP1 scFv and CD3 scFv can be determined using an ELISA assay over a range of concentrations (e.g., lOOnM - 15 pM).
  • Anti-TYRPl and anti-CD3 antibodies can be used as internal reference markers.
  • bispecific scFv-C’ dots on TYRP1+ and TYRP1- AM and CM cell lines can be analyzed after incubating with anti-human IgG 647 (Quantum Simply Cellular) using flow cytometry. Briefly, bispecific scFv-Cy5-C’ dots constructs, can be added to tumor cells and incubated for 30 minutes at 4°C. Following incubation, cells and beads can be washed, resuspended in 200pL cold PBS supplemented with 1% filtered FBS, and analyzed by flow cytometry.
  • TYRP1 receptor numbers per cell for each AM and CM cell line can be quantified using standard maximum fluorescence intensity (MFI) measurements for an Alexa 647 bead set.
  • MFI maximum fluorescence intensity
  • T cell activation can be assessed using a luciferase reporter bioassay, a human (h) CD3- expressing Jurkat cell line stably transfected with a plasmid consisting of a Nuclear Factor of Activated T cell (NFAT) response element and luciferase reporter (Jurkat/NFAT-Luc; Qiagen).
  • Serial dilutions (lOOnM - 15 pM) of two distinct bispecific scFv-C’ dot constructs can be added to Jurkat/NFAT-Luc cells (50,000 cells/well).
  • hCD3-mediated NFAT activation can be evaluated after adding One-Gio luciferase substrate (Promega) for obtaining luminescence readouts with a plate reader (Perkin Elmer).
  • Jurkat/NFAT-Luc cells can be stained with carboxyfluorescein succinimidyl ester (CFSE, ThermoFisher) to assess T cell proliferative capacity after the addition of bispecific C’ dot constructs (e.g., as in I.b.3).
  • CFSE carboxyfluorescein succinimidyl ester
  • To differentiate T cell subsets cells can be stained with anti-CD3, -CD4, and -CD8 antibodies (Invitrogen) for analysis by flow cytometry.
  • T cell-dependent cellular cytotoxicity TDCC
  • T cells can be isolated from leukopaks using methods described in I.b.2.
  • AM and CM cells IxlO 4 cells/well
  • bispecific scFv-C’ dot constructs 37° C, 24 h
  • isolated T cells can be added at an E:T ratio of 10: 1 for an additional 24 h.
  • Time-dependent cellular viability i.e., percentage (%) of cell death relative to controls
  • Non-targeting C’ dots and TYRP1/CD3 can serve as controls.
  • Pilot data shows significantly decreased OC cell viability when treated with combination of 15pM C’ dots and MUC16-targeting CAR T cells, compared to non-treated controls.
  • lead bispecific scFv-C’ dot constructs can be screened for their ability to synergize with CAR T cells to maximize cytotoxicity.
  • Cellular viability can be monitored in real time using procedures in l.b.6 that maximize cytotoxicity, after exposing luc+ AM cells to lead bispecific scFv-C’ dot constructs, and then adding TYRP1 CAR T cells (instead of isolated T cells).
  • Multiple effector-to-target cell ratios can be examined (10: 1, 5: 1, 1 : 1, 0:1).
  • Non-targeting C’ dots, TYRP1/CD3, and CD19-CAR T cells can serve as controls.
  • Cultured tumor and T cell populations demonstrating maximum cytotoxicity after exposure to bispecific scFv-C’ dot constructs can be evaluated by bulk RNAseq to identify functional classes of genes that induce upregulation of expression signatures related to T-cell activation (e.g., 4-1-BB, CD25, CD69), antigen presentation (e.g., Tapi, Tap2), iron metabolism (e.g., Fthl, Tfirc, Slc40al), and cytokines (e.g., Ifnbl, Ifng).
  • T-cell activation e.g., 4-1-BB, CD25, CD69
  • antigen presentation e.g., Tapi, Tap2
  • iron metabolism e.g., Fthl, Tfirc, Slc40al
  • cytokines e.g., Ifnbl, Ifng
  • Candidates exhibiting favorable transcription profiles can be statistically compared and further examined. Changes in expression profiles related to immunostimulatory or immunosuppressive cytokines can be measured in co-culture assays (I.b.5-I.b.7) using a multiplexed cytokine release assay which analyzes up to 12 cytokines/chemokines (LEGENDplexTM, BioLegend®). Cytokine levels in bispecific scFv-C’ dot-exposed samples can be normalized to non-particle-exposed samples.
  • Lead bispecific C’ dot probes can be selected for in vivo PK studies per the following criteria as shown in FIG. 1: (i) (IC50, specificity: BiTE-C’ dot, scFvs-C’ dot > BiTE, scFv alone, (ii) cytotoxicity > 80%.
  • particle attachment of BiTEs or dual scFvs and progressive increases in ligand density per particle can promote increased targeted delivery, cellular engagement, and internalization over that of free BiTEs or scFvs.
  • Particle surface designs can be iterated until maximize binding affinity are achieved.
  • Other biological properties may be improved in addition to binding affinity.
  • TYRP1/GITR which targets a different tumor antigen to that of the CAR T cell therapy, can serve as an alternative bispecific targeting platform to TYRP1/CD3.
  • 2xl0 6 human luc+ AM cells e.g., as described in I.b.1
  • 2xl0 6 human luc+ AM cells can be implanted subcutaneously in the flanks of 6-8-week-old NCG mice, $ and , followed 5 days later by i.v. -injection of IxlO 7 human PBMCs (peripheral blood mononuclear cells), HLA- matched to specific human AM cell lines.
  • Targeted particle and control constructs can be i.v. -injected 3 days after PBMCs (e.g., as shown in FIG. 2).
  • Zirconium-89 (89Zr) radiolabeling of four DFO-conjugated constructs i.e., two 89Zr- DFO-TYRP1/CD3-C’ dots, 89Zr-DFO-C’ dots, and 89Zr-DFO-BiTE
  • Lead C’ dot probes can be selected for therapeutic studies using the following criteria as shown in FIG. 2: (i) specific activity (particle tracer) >1 x 10 4 Ci/mol and % max tumor uptake > 10 %ID/g.
  • a goal of the present set of studies is to evaluate lead bispecific scFv-C’ dot candidates, screened using criteria as set forth in II.a.2 and shown in FIG. 1, in one or more humanized TYRP1+ AM flank tumor models, with and without TA99-CAR T cells. Another goal is to determine whether such combinatorial strategies lead to improved safety profiles and confer a survival benefit over controls in humanized AM models. In addition, the extent to which particle candidates, with and without TA99-CAR T cells, modulate/activate T cell populations and promote inflammatory phenotypes over time can be evaluated.
  • Tumor growth can be monitored every other day by BLI and calipers to assess volumes (length x width x height). Animals can be euthanized for signs of distress, lethargy, body weight changes (>15%), or when tumor volumes reach 2,500mm 3 .
  • Tumor tissues can be harvested in a time-dependent manner, incorporating times of maximal tracer uptake (II.a.2) and endpoints for vehicle-ed animals to assess particle- and non-particle-treated tumor weights, limited IP (Table 1) and histopathology (H&E, IHC for TYRP1 expression).
  • RNA-seq can also be conducted to investigate time-dependent transcriptome-wide alterations in multiple functional processes, such as immune-related responses.
  • Anti -tumor responses can be compared with controls (Schema 3), namely the CAR T cell therapy arm with the same dose schedule as the combination arm, BiTE plus TA99-CAR T cells, and saline vehicle. Histopathology can be performed at the study endpoint as in II.b.2.
  • Lead C’ dot probes can be selected for use in genetically engineered mouse (GEM) models of AM and CM (Stage III) using the following criteria as shown in FIG.
  • GEM genetically engineered mouse
  • a combination strategy that employs a lead TYRP1/CD3 scFv-C’ dot can show a superior therapeutic efficacy in humanized AM models as compared to monotherapies and combinatorial controls.
  • An alternative bispecific construct, TYRP1/GITR, synthesized in Stage I, which targets a different immune cell population can be used if the TYRP1/CD3 scFv-C’ dot is not effective.
  • the combination can promote prolonged survival, reduced off-target effects, and enhanced T cell activity (Table 2) relative to controls at significantly lower CAR T cell doses (0.5 million) relative to that reported previously in AM models, and as was used in prior work (data not shown). If significant anti-tumor cytotoxic responses are not found at doses as described in the present example, progressively higher CAR T cell doses can be used until enhanced efficacy over controls is observed. BiTEs are not expected to exhibit properties favorable for combination strategies given their relatively poorer PK (pharmacokinetics) and short plasma lifetimes ( ⁇ 2 h) relative to those of immunotherapies and nanoparticles described herein. This aspect can be assessed in PET studies (e.g., as described in II.
  • the extent to which the lead bispecific scFv-C’ dot modulates the TME and promotes inflammatory phenotypes in both models with and without TA99- CAR T cells can be evaluated.
  • combinatorial strategies can enhance efficacy, limit suppression, and minimize off-target toxicity observed in humanized AM models as compared to non-targeting C’ dots.
  • Site-specific melanoma induction can be achieved by topically treating the ridges of 6-
  • FPs 10-week-old DBP murine footpads daily with 4-hydroxytamoxifen (4-OHT), followed by scratch wound generation two weeks later. Tumors can form at about 21-30 days after 4-OHT application and wounding.
  • Single-cell transcriptomics (scRNA sequencing, scRNA-seq) can be conducted to investigate transcriptional changes and identify specific cell populations responsive to particle treatment, such as lineage differentiation of Tregs and polarization of macrophages.
  • scRNA-seq both AM and CM murine models can be evaluated.
  • Multiplex IF staining can be performed to detect immune (CD8, CD4, F4/80, FoxP3, PD-L1) and tumor cell proteins (MART-1), with DAPI counterstaining for nuclear detection (Opal 6-plex Detection kit, Akoya Biosciences).
  • Ill.b Investigate contributions of particle-driven cytotoxic and immunostimulatory responses to e fficacy, with and without TA99-CAR T cells.
  • Procedures in II.b.3 and II.b.4 can be used for evaluating growth inhibition and survival in AM and CM mice using the same single lead bispecific ScFv-C’ dot, controls, and combinatorial dose schedules specified in II.b.4.
  • Procedures described in III.a.2 can be used to monitor tumor growth and conduct the same histological analyses.
  • the lead bispecific ScFv-C’ dot candidate that meets the following criteria as shown in FIG. 1 can be selected for product translation: (i) Efficacy: CAR T + bispecific ScFv-C’ dot (combo) > CAR T +/- BiTE; (ii) ) cytotoxic T cell activity: CD8+/TreglOO: (combo>CAR T+/-BiTE); (iii)
  • Humanized luc+ AM models will be created using the NOD SCID gamma (NSG) variant, NCG (NOD Prkdcem26Cd52I12rgem26Cd22/NjuCrl; Gempharmatech, Ltd), to promote efficient and prolonged engraftment of human PBMCs (see I.b.2) relative to NSG mice.
  • NSG NOD SCID gamma
  • Dct-CreER-GFP The GEM AM murine model, B6.Cg-Tg(Dct-rtTA2S*M2) Tg(tetO-HISTlH2BJ/GFP), termed Dct-CreER-GFP, designed to target the tetracycline/doxycy cline-inducible green fluorescent protein (GFP) protein to melanocytes.
  • GFP green fluorescent protein
  • the present example describes that engineered immunomodulatory particle compositions and immunotherapies described herein are able to "switch” on/off the regulatory T cell (Treg) population in the tumor microenvironment. See, for example, as described by U.S. Provisional Application No. 63/542,564 (“Immunomodulatory Nanoparticle Conjugates and Uses Thereof,” filed October 5, 2023) by Bradbury et al., which is incorporated by reference herein in its entirety.
  • immunomodulatory particle compositions comprising a plurality of immune cell engager targeting ligands can be engineered to include 1) a first cellular binding moiety that targets an immune cell-specific moiety (e.g., CD3, e.g., CD8, e.g., a pattern recognition receptor (PRR)); and (2) a second cellular binding moiety that targets antigens expressed by tumor cells (e.g., melanoma cells, e.g., rare melanoma, e.g., acral melanoma) and/or proteins expressed in inflammatory states (e.g., PSMA and/or TYPR1), including neurodegenerative disorders or brain injury (e.g., radiation, trauma) or autoimmune disease.
  • a first cellular binding moiety that targets an immune cell-specific moiety (e.g., CD3, e.g., CD8, e.g., a pattern recognition receptor (PRR)
  • a second cellular binding moiety that targets antigens expressed by
  • engineered cells e.g., engineered immune cells, e.g., CAR T cells
  • an immune cell-specific target e.g., CD3, e.g., CD8, e.g., a pattern recognition receptor (PRR)
  • a second cellular binding moiety that binds to a disease-associated antigen
  • a disease-associated antigen e.g., a tumor-associated antigen (e.g., PSMA, e.g., TYPR1)
  • an inflammatory-associated antigen e.g., an antigen associated with diabetes
  • a neurological disease-associated antigen e.g., a brain injury- associated antigen, e.g., autoimmune-associated antigen
  • a neurological disease-associated antigen e.g., a brain injury- associated antigen, e.g., autoimmune-associated antigen
  • FIG. 4 demonstrates combinatorial strategies enhance activation of CD8+ T cells and significantly decrease regulatory T cells (Tregs) over monotherapies.
  • FIG. 4 Panel a shows data obtained from CD8+ T cells.
  • FIG. 4 Panels b and d show data obtained from CAR T cells and CXCR3+CAR T cells.
  • FIG. 4 Panel c shows data obtained from Tregs.

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Abstract

Sont divulguées dans les présentes des compositions comprenant des cellules immunitaires modifiées et des nanoparticules immunomodulatrices pour une thérapie ciblée localisée dans le traitement du cancer et d'autres maladies, lésions ou affections. Les cellules immunitaires modifiées comprennent des récepteurs antigéniques chimériques (CAR) qui sécrètent un recruteur de cellules immunitaires bispécifiques (par exemple, des recruteurs de lymphocytes T bispécifiques [« BiTE »], par exemple, un recruteur de macrophages bispécifiques, par exemple, un recruteur de neutrophiles bispécifiques). Les cellules immunitaires modifiées par le recruteur de cellules immunitaires bispécifiques CAR sont combinées à des nanoparticules de silice (avec ou sans les ligands de ciblage de recruteur de cellules immunitaires qui leur sont attachés) pour un traitement combinatoire de la maladie ou de l'affection.
PCT/US2024/041718 2023-08-11 2024-08-09 Immunothérapies à particules à base de cellules immunitaires ultra-petites, compositions de cellules immunitaires et leurs utilisations Pending WO2025038450A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10031134B2 (en) * 2010-04-27 2018-07-24 Ventana Medical Systems, Inc. Antibody-nanoparticle conjugates and methods for making and using such conjugates
WO2018156649A1 (fr) * 2017-02-22 2018-08-30 Flagship Pioneering, Inc. Compositions de molécules de modulateur cellules t (tcm) et utilisation connexe

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10031134B2 (en) * 2010-04-27 2018-07-24 Ventana Medical Systems, Inc. Antibody-nanoparticle conjugates and methods for making and using such conjugates
WO2018156649A1 (fr) * 2017-02-22 2018-08-30 Flagship Pioneering, Inc. Compositions de molécules de modulateur cellules t (tcm) et utilisation connexe

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